Methods for detection of nucleic acid sequences in urine

ABSTRACT

Described are non-invasive methods of detecting the presence of specific nucleic acid sequences as well as nucleic acid modifications and alterations by analyzing urine samples for the presence of transrenal nucleic acids. More specifically, the present invention encompasses methods of detecting specific fetal nucleic acid sequences and fetal sequences that contained modified nucleotides by analyzing maternal urine for the presence of fetal nucleic acids. The invention further encompasses methods of detecting specific nucleic acid modifications for the diagnosis of diseases, such as cancer and pathogen infections, and detection of genetic predisposition to various diseases. The invention specifically encompasses methods of analyzing specific nucleic acid modifications for the monitoring of cancer treatment. The invention further encompasses methods of analyzing specific nucleic acids in urine to track the success of transplanted cells, tissues and organs. The invention also encompasses methods for evaluating the effects of environmental factors and aging on the genome.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part and a division of applicationSer. No. 09/609,162 filed Jul. 3, 2000, now U.S. Pat. No. 6,287,820,which is a continuation-in-part and a division of application Ser. No.09/230,704, filed Feb. 4, 2000, now U.S. Pat. No. 6,251,704, which isthe United States national phase of International Patent Application No.PCT/US98/10965, filed May 29, 1998, which claims priority of U.S.provisional patent applications No. 60/058,170, filed May 30, 1997, andSer. No. 60/048,381, filed Jun. 3, 1997.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

(Not Applicable)

TECHNICAL FIELD

The present invention encompasses non-invasive methods of detecting thepresence of specific nucleic acid sequences as well as nucleic acidmodifications and alterations by analyzing urine samples for thepresence of transrenal nucleic acids. More specifically, the presentinvention encompasses methods of detecting specific fetal nucleic acidsequences and fetal sequences that contained modified nucleotides byanalyzing maternal urine for the presence of fetal nucleic acids. Theinvention further encompasses methods of detecting specific nucleic acidmodifications for the diagnosis of diseases, such as cancer and pathogeninfections, and detection of genetic predisposition to various diseases.The invention specifically encompasses methods of analyzing specificnucleic acid modifications for the monitoring of cancer treatment. Theinvention further encompasses methods of analyzing specific nucleicacids in urine to track the success of transplanted cells, tissues andorgans. The invention also encompasses methods for evaluating theeffects of environmental factors and aging on the genome.

BACKGROUND

Human genetic material is an invaluable source of information. Over thelast several decades, scientific endeavors have developed many methodsof analyzing and manipulating this genetic material (nucleic acids, DNAand RNA) for a variety of uses. These applications of molecular biologyhave been at the heart of numerous modern medical techniques fordiagnosis and treatment. Thus, means of obtaining, isolating andanalyzing this genetic material has become of foremost importance.

Until now, the fragile nature of nucleic acids, and their locationencapsulated within cells, made the acquisition of genetic material fordiagnosis in certain cases necessarily intrusive. For example, tumordiagnosis often requires surgery to obtain tumor cells. Similarly,doctors perform amniocenteses to obtain fetal DNA for a variety ofdiagnostic uses. This procedure requires the insertion of a needlethrough the abdomen of a pregnant woman and into the amniotic sac. Suchintrusive practices carry with them a level of risk to both the fetusand the mother. While developments in ultrasound have contributed lessintrusive alternative methods of fetal monitoring during pregnancy,these methods are not appropriate for diagnosing certain genetic defectsand are not effective during the early stages of pregnancy, even fordetermining fetal sex.

Recent studies into the various mechanisms and consequences of celldeath have opened a potential alternative to the invasive techniquesdescribed above. It is well established that apoptotic cell death isfrequently accompanied by specific internucleosomal fragmentation ofnuclear DNA. However, the fate of these chromatin degradation productsin the organism has not been investigated in detail.

Based on the morphology of dying cells, it is believed that there existtwo distinct types of cell death, necrosis and apoptosis. Kerr, J. F. etal., Br. J. Cancer 26:239-257, (1972). Cell death is an essential eventin the development and function of multicellular organisms. In adultorganisms, cell death plays a complementary role to mitosis in theregulation of cell populations. The pathogenesis of numerous diseasesinvolves failure of tissue homeostasis which is presumed to be linkedwith cytotoxic injury or loss of normal control of cell death. Apoptosiscan be observed during the earliest stages of embryogenesis in theformation of organs, substitution of one tissue by another andresorption of temporary organs.

Necrosis is commonly marked by an early increase in total cell volumeand subcellular organelle volume followed by autolysis. Necrosis isconsidered to be a catastrophic metabolic failure resulting directlyfrom severe molecular and/or structural damage. Apoptosis is anatraumatic programmed cell death that naturally occurs in the normaldevelopment and maintenance of healthy tissues and organs. Apoptosis isa much more prevalent biological phenomenon than necrosis. Kerr, J. F.et al., Br. J. Cancer 26:239-257, (1972). Umansky, S. Molecular Biology(Translated from Molekulyarnaya Biologiya) 30:285-295, (1996). Vaux, D.L. et al., Proc Natl Acad Sci USA. 93:2239-2244, (1996). Umansky, S., J.Theor. Biol. 97:591-602, (1982). Tomei, L. D. and Cope, F. D. Eds.,Apoptosis: The Molecular Basis of Cell Death, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1991).

Apoptosis is also a critical biological function which occurs naturallyduring embryogenesis, positive and negative selection of T andB-lymphocytes, glucocorticoid induced lymphocyte death, death induced byradiation and temperature shifts, and death following deprivation ofspecific growth factors. In addition, apoptosis is an important part ofan organism's defense against viral infection. Apoptosis has beenobserved in preneoplastic foci found in the liver following tumorpromoter phenobarbital withdrawal, in involuting hormone-dependenttissues and in tumors upon hormone withdrawal. Many antitumor drugs,including inhibitors of topoisomerase II as well as tumor necrosisfactors induce apoptotic cell death. Apoptotic cell death ischaracterized by morphologic changes such as cellular shrinkage,chromatin condensation and margination, cytoplasmic blebbing, andincreased membrane permeability. Gerschenson et al. (1992) FASEB J.6:2450-2455; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267-293.Specific internucleosomal DNA fragmentation is a hallmark for many, butnotably not all, instances of apoptosis.

In necrotic cells, DNA is also degraded but as a result of theactivation of hydrolytic enzymes, generally yielding mono- andoligonucleotide DNA products. Afanasyev, V. N. et al., FEBS Letters.194: 347-350 (1986).

Recently, earlier stages of nuclear DNA degradation have been described.It was shown that after pro-apoptotic treatments, DNA cleavage beginswith the formation of high molecular weight DNA fragments in the rangeof 50-300 kilobases, the size of DNA found in chromosome loops. Walker,P. R. et al., Cancer Res. 51:1078-1085 (1991). Brown, D. G. et al., J.Biol. Chem. 268:3037-3039 (1993). These large fragments are normallydegraded to nucleosomes and their oligomers. However, in some cases ofapoptotic cell death only high molecular weight DNA fragments can beobserved. Oberhammer, F. et al., EMBO J. 12:3679-3684 (1993). There arealso data on the appearance of such fragments in some models of necroticcell death. Kataoka, A. et al., FEBS Lett. 364:264-267 (1995).

Available data on the fate of these chromatin degradation products inorganisms provide little guidance. Published results indicate that onlysmall amounts of DNA can be detected in blood plasma or serum. Fournie,G. J. et al., Gerontology 39:215-221 (1993). Leon, S. et al., CancerResearch 37:646-650 (1977). It can be difficult to ensure that this DNAdid not originate from white blood cells as a result of their lysisduring sample treatment.

Extracellular DNA with microsatellite alterations specific for smallcell lung cancer and head and neck cancer was found in human serum andplasma by two groups. Chen, X. Q. et al., Nature Medicine 2:1033-1035(1996). Nawroz, H. et al., Nature Medicine 2:1035-1037 (1996). Othershave proposed methods of detecting mutated oncogene sequences in solubleform in blood. U.S. Pat. No. 5,496,699, to George D. Sorenson. However,the use of blood or plasma as a source of DNA is both intrusive to thepatient and problematic for the diagnostic technician. In particular, ahigh concentration of proteins (about 100 mg/ml) as well as the presenceof compounds which inhibit the polymerase chain reaction (PCR) make DNAisolation and analysis difficult.

A few groups have identified, by PCR, DNA modifications or viralinfections in bodily fluids, including urine. Ergazaki, M., et al.,“Detection of the cytomegalovirus by the polymerase chain reaction, DNAamplification in a kidney transplanted patient,” In Vivo 7:531-4 (1993);Saito, S., “Detection of H-ras gene point mutations in transitional cellcarcinoma of human urinary bladder using polymerase chain reaction,”Keio J Med 41:80-6 (1992). Mao, L., et al., “Molecular Detection ofPrimary Bladder Cancer by Microsatellite Analysis,” Science 271:659-662(1996). The DNA that these groups describe detecting is from kidneycells or cells lining the bladder. When detecting a viral infection,many viruses infect cells of the bladder, thereby obtaining entry intothe urine. The descriptions do not teach methods of detecting DNAsequences in urine that do not originate from the bladder or kidneycells, and thus would not include DNA that passes through the kidneybarrier and remains in detectable form in urine prior to detection.

What is needed is a non-invasive method of obtaining nucleic acidsamples from cells located outside the urinary tract, for use indiagnostic and monitoring applications. The ability to obtain, in anon-invasive way, and analyze specific nucleic acid sequences would havevalue for purposes including, but not limited to, determining the sex ofa fetus in the early stages of development, diagnosing fetal geneticdisorders, and achieving early diagnosis of cancer. The presence of Ychromosome gene sequences in the urine of a pregnant woman would beindicative of a male fetus. The presence of gene sequences specific to acertain type of tumor in the urine of a patient would be a marker forthat tumor. Thus, such methods would be useful in suggesting and/orconfirming a diagnosis.

Methods for analysis of transrenal nucleic acids and are in urine havenot been previously described.

All references cited herein are incorporated by reference in theirentirety.

SUMMARY OF THE INVENTION

The present invention encompasses non-invasive methods of detecting thepresence of specific nucleic acid sequences as well as nucleic acidmodifications and alterations by analyzing urine samples for thepresence of transrenal nucleic acids. More specifically, the presentinvention encompasses methods of detecting specific fetal nucleic acidsequences and fetal sequences that contained modified nucleotides byanalyzing maternal urine for the presence of fetal nucleic acids. Theinvention further encompasses methods of detecting specific nucleic acidmodifications for the diagnosis of diseases, such as cancer and pathogeninfections, and detection of genetic predisposition to various diseases.The invention specifically encompasses methods of analyzing specificnucleic acid modifications for the monitoring of cancer treatment. Theinvention further encompasses methods of analyzing specific nucleicacids in urine to track the success of transplanted cells, tissues andorgans. The invention also encompasses methods for evaluating theeffects of environmental factors and aging on the genome.

The present invention encompasses methods of analyzing a fragment offetal DNA that has crossed the placental and kidney barriers,comprising: obtaining a urine sample, suspected of containing fetalpolymeric transrenal nucleic acids, from a pregnant female; and assayingfor the presence of said fetal polymeric DNA in said urine sample.

The target fetal DNA sequence can be, for example, a sequence that ispresent only on the Y chromosome. The step of assaying for the presenceof unique fetal DNA sequence can be performed using one or more of avariety of techniques, including, but not limited to, hybridization,cycling probe reaction, cleavage product detection, polymerase chainreaction, nested polymerase chain reaction, polymerase chainreaction-single strand conformation polymorphism, ligase chain reaction,strand displacement amplification and restriction fragments lengthpolymorphism. The step of performing the polymerase chain reaction cancomprise using primers substantially complementary to a portion of theunique fetal DNA sequence, and the unique fetal DNA sequence can be asequence that is present in the paternal genome and not present in thematernal genome.

The present invention further encompasses methods having the step ofreducing DNA degradation in the urine sample. Reducing DNA degradationcan be by treatment with compounds selected from the group consistingof: ethylenediaminetetraacetic acid, guanidine-HCl, Guanidineisothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate. DNAdegradation can further be reduced by taking a urine sample that hasbeen held in the bladder less than 12 hours.

The present invention encompasses methods where DNA in the urine sampleis substantially isolated prior to assaying for the presence of a uniquefetal DNA sequence in the urine sample. Substantial isolation can be by,but is not limited to, precipitation and adsorption on a resin.

In one embodiment of the present invention, the presence of theparticular unique fetal DNA sequence is indicative of a genetic disease.

In some cases, it can be desirable to filter the urine sample to removecontaminating nucleic acids before assaying. In a specific embodiment,the filtering removes DNA comprising more than about 1000 nucleotides.

The present invention also encompasses methods of analyzing a targetnucleic acid sequence in urine, comprising: providing a urine sample;and assaying the urine sample for the presence of a target DNA sequencethat has crossed the kidney barrier.

The step of assaying for the presence of a target DNA sequence can beselected from the group consisting of hybridization, cycling probereaction, polymerase chain reaction, nested polymerase chain reaction,polymerase chain reaction-single strand conformation polymorphism,ligase chain reaction, strand displacement amplification and restrictionfragments length polymorphism. The step of assaying for the presence ofa target DNA sequence can comprise techniques for amplifying the targetDNA.

In one embodiment, the target DNA sequence comprises an altered genesequence, and that altered gene sequence can comprise a modificationoccurring in tumor cells in specific.

The present invention further encompasses methods having the step ofreducing DNA degradation in the urine sample prior to assaying the urinesample for the presence of a target DNA sequence that has crossed thekidney barrier. Reducing DNA degradation can be by treatment withcompounds selected from the group consisting of:ethylenediaminetetraacetic acid, guanidine-HCl, Guanidineisothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate. DNAdegradation can further be reduced by taking a urine sample that hasbeen held in the bladder less than 12 hours.

The present invention encompasses methods where DNA in the urine sampleis substantially isolated prior to assaying for the presence of a targetDNA sequence that has crossed the kidney barrier. Substantial isolationcan be by, but is not limited to, precipitation and adsorption on aresin.

In some cases, it is desirable to filter the urine sample to removecontaminating nucleic acids before assaying for the presence of a targetDNA sequence that has crossed the kidney barrier. In a specificembodiment, the filtering removes DNA comprising more than about 1000nucleotides.

The present invention also encompasses methods of analyzing a targetnucleic acid sequence in urine, comprising: providing a urine sample,suspected of containing DNA that has crossed the kidney barrier, from apatient; amplifying a target DNA sequence in the DNA that has crossedthe kidney barrier, comprising using a primer substantiallycomplementary to a portion of the target DNA sequence that does notoccur in cells of the urinary tract of the patient, to make amplifiedtarget DNA; and detecting the presence of the amplified target DNA.Amplification can comprise performing a polymerase chain reaction. Thetarget DNA sequence can comprise an altered gene sequence, such as amodification occurring in tumor cells.

The present invention further encompasses methods having the step ofreducing DNA degradation in the urine sample prior to amplifying atarget DNA sequence in the DNA that has crossed the kidney barrier.Reducing DNA degradation can be by treatment with compounds selectedfrom the group consisting of: ethylenediaminetetraacetic acid,guanidine-HCl, Guanidine isothiocyanate, N-lauroylsarcosine, andNa-dodecylsulphate. DNA degradation can further be reduced by taking aurine sample that has been held in the bladder less than 12 hours.

The present invention encompasses methods where DNA in the urine sampleis substantially isolated prior to amplifying a target DNA sequence inthe DNA that has crossed the kidney barrier. Substantial isolation canbe by, but is not limited to, precipitation and adsorption on a resin.

In some cases, it can be desirable to filter the urine sample to removecontaminating nucleic acids before amplifying a target DNA sequence inthe DNA that has crossed the kidney barrier. In a specific embodiment,filtering removes DNA comprising more than about 1000 nucleotides.

The present invention further encompasses a method of determining thesex of a fetus, comprising: obtaining a urine sample, suspected ofcontaining fetal male DNA, from a pregnant female; amplifying a portionof the male DNA present in the urine sample by the polymerase chainreaction, using an oligodeoxynucleotide primer having sequences specificto a portion of the Y chromosome, to produce amplified DNA; anddetecting the presence of the amplified DNA.

The present invention encompasses a diagnostic kit for detecting thepresence of human male fetal DNA in maternal urine, comprising: reagentsto facilitate the isolation of DNA from urine; reagents to facilitateamplification of DNA by the polymerase chain reaction; a heat stable DNApolymerase; and an oligodeoxynucleotide specific for a sequence onlyoccurring on the Y chromosome.

Additionally, the present invention encompasses oligonucleotide primersfor the amplification of sequences of the Y chromosome, comprising SEQID NO: 3 and SEQ ID NO: 4. A kit for detecting male nucleic acid is alsoencompasses, this pair of primers. The invention also encompasses amethod for detecting Y-chromosome nucleic acid, comprising: carrying outa polymerase chain reaction using these primers and detecting amplifiedY-chromosome nucleic acid.

Oligonucleotide probes are also disclosed, including SEQ ID NO: 3 andSEQ ID NO: 4, which can be used for the detection of male nucleic acid.

The present invention further encompasses methods of detecting cancer ina patient, comprising: providing a urine sample from a patient; andanalyzing said urine sample for a nucleic acid sequence, indicative ofcancer, that has crossed the kidney barrier. In a specific embodiment,said step of analyzing for the presence of said nucleic acid sequence isselected from the group consisting of hybridization, cycling probereaction, polymerase chain reaction, nested polymerase chain reaction,polymerase chain reaction-single strand conformation polymorphism,ligase chain reaction, strand displacement amplification and restrictionfragments length polymorphism. In another embodiment, analyzing for thepresence of said nucleic acid sequence comprises amplifying said nucleicacid sequence indicative of cancer.

In another specific embodiment, said analyzing comprises quantifying thenumber of copies of said nucleic acid sequence.

In one embodiment said nucleic acid sequence contains an anomalyindicative of colon cancer. In another embodiment, said nucleic acidsequence contains mutant K-ras DNA.

It is helpful in some embodiments to include a step to reduce DNAdegradation in said urine sample, which in one embodiment encompassestreatment with a compound selected from the group comprising:ethylenediaminetetraacetic acid, guanidine-HCl, Guanidineisothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate.

In another embodiment the urine sample has been held in the bladder lessthan 12 hours.

In one embodiment, it is beneficial to substantially isolate saidnucleic acid sequence prior to assaying the urine for the presence of anucleic acid sequence, indicative of cancer, that has crossed the kidneybarrier. In alternate embodiments, the nucleic acid sequence issubstantially isolated by precipitation or by treatment with a solidadsorbent material. In another embodiment, the urine sample is filteredto remove contaminants, and, in a specific embodiment, the filteringremoves DNA comprising more than about 1000 nucleotides.

Also encompassed by the present invention is a method of monitoringtransplanted material in a patient, comprising: providing a urine samplesuspected of containing nucleic acid from transplanted material; andanalyzing said urine sample for a nucleic acid sequence that has crossedthe kidney barrier and that was not present in the patient prior totransplantation. In a specific embodiment, the nucleic acid sequence isnot present in cells of the urinary tract of said patient.

In a specific embodiment, the analyzing comprises amplifying saidnucleic acid sequence with a primer substantially complementary to apart of said nucleic acid sequence that does not occur in cells of theurinary tract of the patient, to make amplified target DNA, anddetecting the presence of said amplified target DNA. More specifically,the amplifying can comprise performing a polymerase chain reaction.

In another specific embodiment is included the additional step ofreducing DNA degradation in said urine sample, which can be performed inany way known, but, without limitation, includes situations whereinreducing DNA degradation is by treatment with a compound selected fromthe group consisting of: ethylenediaminetetraacetic acid, guanidine-HCl,Guanidine isothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate.

In some embodiments, said urine sample has been held in the bladder lessthan 12 hours.

It is desirable in some embodiments to substantially isolate saidnucleic acid sequence. In alternate embodiments, the nucleic acidsequence is substantially isolated by precipitation, and/or byadsorption on a resin.

Additionally, one can filter the urine sample to remove contaminants. Ina specific embodiment, this filtering removes DNA comprising more thanabout 1000 nucleotides.

In yet another embodiment a method of monitoring cancer treatment in apatient is encompassed, comprising: providing a urine sample from apatient; and analyzing said urine sample for the quantity of a nucleicacid sequence, indicative of cancer, that has crossed the kidneybarrier.

Further encompassed by the present invention is a diagnostic kit fordetecting a genetic mutation indicative of cancer in the DNA of apatient, comprising: reagents to facilitate the isolation of DNA fromurine; reagents to facilitate amplification of DNA by the polymerasechain reaction; a heat stable DNA polymerase; and anoligodeoxynucleotide specific for a sequence only occurring in a geneticmutation characteristic of cancer.

Also encompassed by the present invention is a diagnostic kit fordetecting DNA from a transplanted material in the urine of a patient,comprising: reagents to facilitate the isolation of DNA from urine;reagents to facilitate amplification of DNA by the polymerase chainreaction; a heat stable DNA polymerase; and an oligodeoxynucleotidespecific for a sequence that occurs in the transplanted material, anddid not occur in the patient prior to transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph of an agarose gel, stained with ethidiumbromide, which depicts the detection of polymeric DNA in urine samplestaken from mice injected with λ phage DNA. The results of twoexperiments (lanes 1 and 2) are presented.

FIG. 1B is an autoradiograph of an agarose gel which depicts thedetection of 32P-labeled λ phage DNA in the urine from mice injectedwith phage DNA. The results of two experiments (lanes 1 and 2) arepresented.

FIG. 2 is a photograph of an agarose gel which depicts the detection bygel electrophoresis of human Raji lymphoma cell DNA sequences from theurine of mice preinoculated with irradiated human cells. Lanes: 1—urineDNA from control mouse; 2—control human DNA; 3—urine DNA from mouse thatwas injected with human cells.

FIG. 3 is a photograph of an agarose gel which depicts the detection ofY chromosome specific sequences of DNA from the urine of a woman who hadbeen transfused with blood from a male 10 days earlier. Lanes: 1—markers(pBR322 DNA-MspI digest); 2—positive control (0.1 μg of total DNA fromlymphocytes of a male donor); 3—blank sample (salt solution passedthrough all the procedures of DNA isolation and analysis); 4—negativecontrol (no added DNA); 5—urine DNA after blood transfusion.

FIG. 4A is a photograph of an agarose gel which depicts the detection,in the urine of pregnant women carrying male fetuses, of a 154 base pairfragment of the Y chromosome-specific repeated DNA sequence. Lanes:M-molecular weight standard; 1—negative control (no DNA added);2-5—positive controls (0.1, 1.0, 10 and 100 pg of total male DNA,respectively); 6 and 8—male fetuses; 7—female fetus; 9—blank sample;10—urine DNA from non-pregnant woman.

FIG. 4B is a photograph of an agarose gel which depicts the detection,in the urine of pregnant women carrying male fetuses, of a 97 base pairfragment of the Y chromosome-specific repeated DNA sequence. Lanes:M-molecular weight standard; 1-3—positive controls (0.1, 1.0 and 10 pgof male total DNA, respectively); 4 and 5—female fetuses; 6 and 7—malefetuses; 8—blank sample; 9—urine DNA from non-pregnant woman.

FIG. 5 is a photograph of an agarose gel which depicts the detection ofa Y chromosome-specific single-copy DNA sequence (198 base pairs) in theurine of pregnant women carrying male fetuses. Lanes: M-molecular weightstandard; 1—negative control (no DNA added); 2-5—positive controls (1,10, 100 and 1000 pg of total male DNA, respectively); 6 and 7—malefetuses; 8—female fetus; 9—blank sample; 10—urine DNA from non-pregnantwoman.

FIG. 6 is a photograph of an agarose gel which depicts the kinetics ofDNA degradation over time as a result of endogenous DNase activity inurine, wherein the lanes contain the following: Lane 1—positive control(200 pg of λ phage DNA added to PCR tube); Lanes 2-5—samples incubatedfor 0, 30 min., 60 min. and 120 min., respectively.

FIG. 7 is an autoradiograph of a Zeta-probe membrane which depicts thedetection, by hybridization, of specific Y chromosome DNA sequences inurine samples from pregnant women. Lanes: 1—negative control(non-pregnant female); 2—positive control (male total genome DNA, 5 ng);3,4—male fetuses; 5,6—female fetuses.

FIGS. 8A, 8B, and 8C are photographs of agarose gels which compare fetalDNA to maternal urine DNA at gestation ages of approximately 7-8 weeks.FIG. 8A represents fetal DNA, FIG. 8B represents maternal urine DNAprepared by simple 10—fold urine dilution, and FIG. 8C representsmaternal urine DNA prepared by GEAE Sephadex A-25 adsorption. M-male;f-female.

FIG. 9 is a photograph of an agarose gel showing the effect on PCR ofthe adsorption of urine DNA on Hybond N filters under variousconditions. Lanes 1-4-20 fg, 1 pg, 2 pg or 10 pg male DNA were added per1 μof female urine. Control - 10 μl aliquots of 10-fold diluted urinewere taken directly into PCR tubes. Other urine samples were made highlyconcentrated in salt (10×SSC) or alkaline (adjusted to pH 12 with NaOH)and handled with “filter transfer” method. nc- negative control;m-molecular weight standard.

FIGS. 10A, 10B, and 10C are photographs of an agarose gel showing theeffect of the adsorption of urine DNA using Hybond N filters on DNAdegradation. A-control (lanes: 1-10 μl aliquot of 10-fold diluted urinewas taken directly into PCR tubes just after male DNA addition; 2-10 μlaliquot of 10-fold diluted urine was taken directly into PCR tubes afterincubation overnight at room temperature; 3-Hybond N filter wasincubated in urine overnight and used for DNA filter transfer (10 μlaliquot of eluate from filter was used for the analysis). B—all theprocedures as in A, except the urine samples were made 10 mM in EDTA.C—all the procedures as in A, except the urine samples were made 10 mMin EDTA and adjusted to pH 12.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the new discovery that geneticmaterial from cells in the body can pass through the kidney barrier andappear in the urine of a mammal in a form sufficiently intact to beanalyzed. In addition, genetic material from cells of the developingembryo can cross both the placental and kidney barriers and appear inthe pregnant mother's urine. The present invention encompassesnon-invasive methods of detecting the presence of specific nucleic acidsequences as well as nucleic acid modifications and alterations byanalyzing urine samples for the presence of transrenal nucleic acids.More specifically, the present invention encompasses methods ofdetecting specific fetal nucleic acid sequences and fetal sequences thatcontained modified nucleotides by analyzing maternal urine for thepresence of fetal nucleic acids. The invention further encompassesmethods of detecting specific nucleic acid modifications for thediagnosis of diseases, such as cancer and pathogen infections, anddetection of genetic predisposition to various diseases. The inventionspecifically encompasses methods of analyzing specific nucleic acidmodifications for the monitoring of cancer treatment. The inventionfurther encompasses methods of analyzing specific nucleic acids in urineto track the success of transplanted cells, tissues and organs. Theinvention also encompasses methods for evaluating the effects ofenvironmental factors and aging on the genome.

This invention further encompasses novel primers, YZ1 and YZ2, for usein amplification techniques of the present invention, as set forth inExample 3, below.

The methods of the present invention offer improvements over previousmethods of diagnosis, detection and monitoring due to their inherentlynon-invasive nature.

To facilitate understanding of the invention, a number of terms aredefined below.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the transcription of an RNA sequence. Theterm “genome” refers to the complete gene complement of an organism,contained in a set of chromosomes in eukaryotes.

A “wild-type” gene or gene sequence is that which is most frequentlyobserved in a population and is thus arbitrarily designed the “normal”or “wild-type” form of the gene. In contrast, the term “modified”,“mutant”, “anomaly” or “altered” refers to a gene, sequence or geneproduct which displays modifications in sequence and or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene, sequence or gene product. For example, an alteredsequence detected in the urine of a patient can display a modificationthat occurs in DNA sequences from tumor cells and that does not occur inthe patient's normal (i.e. non cancerous) cells. It is noted thatnaturally-occurring mutants can be isolated; these are identified by thefact that they have altered characteristics when compared to thewild-type gene or gene product. Without limiting the invention to thedetection of any specific type of anomaly, mutations can take manyforms, including addition, addition-deletion, deletion, frame-shift,missense, point, reading frame shift, reverse, transition andtransversion mutations as well as microsatellite alterations.

A “disease associated genetic anomaly” refers to a gene, sequence orgene product that displays modifications in sequence when compared tothe wild-type gene and that is indicative of the propensity to developor the existence of a disease in the carrier of that anomaly. A diseaseassociated genetic anomaly encompasses, without limitation, inheritedanomalies as well as new mutations.

The term “unique fetal DNA sequence” is defined as a sequence of nucleicacids that is present in the genome of the fetus, but not in thematernal genome.

The terms “oligonucleotide” and “polynucleotide” and “polymeric” nucleicacid are interchangeable and are defined as a molecule comprised of twoor more deoxyribonucleotides or ribonucleotides, preferably more thanthree, and usually more than ten. The exact size will depend on manyfactors, which in turn depends on the ultimate function or use of theoligonucleotide. The oligonucleotide can be generated in any manner,including chemical synthesis, DNA replication, reverse transcription, ora combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also can be said to have 5′ and 3′ ends.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former can be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” can occur naturally, as in a purified restriction digest or beproduced synthetically.

A primer is selected to be “substantially” complementary to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment can beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

A “target” nucleic acid is a nucleic acid sequence to be evaluated byhybridization, amplification or any other means of analyzing a nucleicacid sequence, including a combination of analysis methods.

“Hybridization” methods involve the annealing of a complementarysequence to the target nucleic acid (the sequence to be analyzed). Theability of two polymers of nucleic acid containing complementarysequences to find each other and anneal through base pairing interactionis a well-recognized phenomenon. The initial observations of the“hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960)have been followed by the refinement of this process into an essentialtool of modern biology. Hybridization encompasses, but is not limitedto, slot, dot and blot hybridization techniques.

It is important for some diagnostic applications to determine whetherthe hybridization represents complete or partial complementarity. Forexample, where it is desired to detect simply the presence or absence ofpathogen DNA (such as from a virus, bacterium, fungi, mycoplasma,protozoan) it is only important that the hybridization method ensureshybridization when the relevant sequence is present; conditions can beselected where both partially complementary probes and completelycomplementary probes will hybridize. Other diagnostic applications,however, could require that the hybridization method distinguish betweenpartial and complete complementarity. It may be of interest to detectgenetic polymorphisms.

Methods that allow for the same level of hybridization in the case ofboth partial as well as complete complementarity are typically unsuitedfor such applications; the probe will hybridize to both the normal andvariant target sequence. The present invention contemplates that forsome diagnostic purposes, hybridization be combined with othertechniques (such as restriction enzyme analysis). Hybridization,regardless of the method used, requires some degree of complementaritybetween the sequence being analyzed (the target sequence) and thefragment of DNA used to perform the test (the probe). (Of course, onecan obtain binding without any complementarity but this binding isnonspecific and to be avoided.)

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Specific bases not commonly found innatural nucleic acids can be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes can containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

As used herein, the term “Tm” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the Tm ofnucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the Tm value can be calculated by theequation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridisation, in Nucleic Acid Hybridisation (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of Tm.

The term “probe” as used herein refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, which forms a duplexstructure or other complex with a sequence in another nucleic acid, dueto complementarity or other means of reproducible attractiveinteraction, of at least one sequence in the probe with a sequence inthe other nucleic acid. Probes are useful in the detection,identification and isolation of particular gene sequences. It iscontemplated that any probe used in the present invention will belabeled with any “reporter molecule,” so that it is detectable in anydetection system, including, but not limited to, enzyme (e.g., ELISA, aswell as enzyme-based histochemical assays), fluorescent, radioactive,and luminescent systems. It is further contemplated that theoligonucleotide of interest (i.e., to be detected) will be labeled witha reporter molecule. It is also contemplated that both the probe andoligonucleotide of interest will be labeled. It is not intended that thepresent invention be limited to any particular detection system orlabel.

The term “label” as used herein refers to any atom or molecule which canbe used to provide a detectable (preferably quantifiable) signal, andwhich can be attached to a nucleic acid or protein. Labels providesignals detectable by any number of methods, including, but not limitedto, fluorescence, radioactivity, colorimetry, gravimetry, X-raydiffraction or absorption, magnetism, and enzymatic activity.

The term “substantially single-stranded” when used in reference to anucleic acid target means that the target molecule exists primarily as asingle strand of nucleic acid in contrast to a double-stranded targetwhich exists as two strands of nucleic acid which are held together byinter-strand base pairing interactions.

The term “sequence variation” as used herein refers to differences innucleic acid sequence between two nucleic acid templates. For example, awild-type structural gene and a mutant form of this wild-type structuralgene can vary in sequence by the presence of single base substitutionsand/or deletions or insertions of one or more nucleotides. These twoforms of the structural gene are said to vary in sequence from oneanother. A second mutant form of the structural gene can exit. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

The terms “structure probing signature,” “hybridization signature” and“hybridization profile” are used interchangeably herein to indicate themeasured level of complex formation between a target nucleic acid and aprobe or set of probes, such measured levels being characteristic of thetarget nucleic acid when compared to levels of complex formationinvolving reference targets or probes.

“Oligonucleotide primers matching or complementary to a gene sequence”refers to oligonucleotide primers capable of facilitating thetemplate-dependent synthesis of single or double-stranded nucleic acids.Oligonucleotide primers matching or complementary to a gene sequence canbe used in PCRs, RT-PCRs and the like.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which can be single- ordouble-stranded, and represent the sense or antisense strand.

A “deletion” is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, are absent.

An “insertion” or “addition” is that change in a nucleotide or aminoacid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to,naturally occurring sequences.

A “substitution” results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

A “modification” in a nucleic acid sequence refers to any change to anucleic acid sequence, including, but not limited to a deletion, anaddition, an addition-deletion, a substitution, an insertion, areversion, a transversion, a point mutation, a microsatilite alteration,methylation or nucleotide adduct formation.

As used herein, the terms “purified”, “decontaminated” and “sterilized”refer to the removal of contaminant(s) from a sample.

As used herein, the terms “substantially purified” and “substantiallyisolated” refer to nucleic acid sequences that are removed from theirnatural environment, isolated or separated, and are preferably 60% free,more preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An “isolatedpolynucleotide” is therefore a substantially purified polynucleotide. Itis contemplated that to practice the methods of the present inventionpolynucleotides can be, but need not be substantially purified. Avariety of methods for the detection of nucleic acid sequences inunpurified form are known in the art.

“Amplification” is defined as the production of additional copies of anucleic acid sequence and is generally carried out using polymerasechain reaction or other technologies well known in the art (e.g.,Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y. [1995]). As used herein, the term“polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis(U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated byreference), which describe a method for increasing the concentration ofa segment of a target sequence in a mixture of genomic DNA withoutcloning or purification. This process for amplifying the target sequenceconsists of introducing a large excess of two oligonucleotide primers tothe DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified”.

As used herein, the term “polymerase” refers to any enzyme suitable foruse in the amplification of nucleic acids of interest. It is intendedthat the term encompass such DNA polymerases as Taq DNA polymeraseobtained from Thermus aquaticus, although other polymerases, boththermostable and thermolabile are also encompassed by this definition.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level that can be detected by severaldifferent methodologies (e.g., staining, hybridization with a labeledprobe; incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of 32P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular, theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications. Amplified targetsequences can be used to obtain segments of DNA (e.g., genes) forinsertion into recombinant vectors.

As used herein, the terms “PCR product” and “amplification product”refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity canbe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there can be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids.

The term “homology” refers to a degree of complementarity. There can bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence can be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding can be tested by the use of a second target whichlacks even a partial degree of complementarity (e.g., less than about30% identity); in the absence of non-specific binding the probe will nothybridize to the second non-complementary target.

Numerous equivalent conditions can be employed to comprise either low orhigh stringency conditions; factors such as the length and nature (DNA,RNA, base composition) of the probe and nature of the target (DNA, RNA,base composition, present in solution or immobilized, etc.) and theconcentration of the salts and other components (e.g., the presence orabsence of formamide, dextran sulfate, polyethylene glycol) areconsidered and the hybridization solution can be varied to generateconditions of either low or high stringency hybridization differentfrom, but equivalent to, the above listed conditions. The term“hybridization” as used herein includes “any process by which a strandof nucleic acid joins with a complementary strand through base pairing”(Coombs, Dictionary of Biotechnology, Stockton Press, New York N.Y.[1994].

“Stringency” typically occurs in a range from about Tm-5° C. (5° C.below the Tm of the probe) to about 20° C. to 25° C. below Tm. As willbe understood by those of skill in the art, a stringent hybridizationcan be used to identify or detect identical polynucleotide sequences orto identify or detect similar or related polynucleotide sequences.

As used herein the term “hybridization complex” refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds can be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex can be formed in solution (e.g., C0t or R0tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized to a solid support (e.g., anylon membrane or a nitrocellulose filter as employed in Southern andNorthern blotting, dot blotting or a glass slide as employed in in situhybridization, including FISH [fluorescent in situ hybridization]).

As used herein, the term “antisense” is used in reference to RNAsequences which are complementary to a specific RNA (e.g., mRNA) or DNAsequence. Antisense RNA can be produced by any method, includingsynthesis by splicing the gene(s) of interest in a reverse orientationto a viral promoter which permits the synthesis of a coding strand. Onceintroduced into a cell, this transcribed strand combines with naturalmRNA produced by the cell to form duplexes. These duplexes then blockeither further transcription of the mRNA or its translation. In thismanner, mutant phenotypes can be generated. The term “antisense strand”is used in reference to a nucleic acid strand that is complementary tothe “sense” strand. The designation (−) (i.e., “negative”) is sometimesused in reference to the antisense strand, with the designation (+)sometimes used in reference to the sense (i.e., “positive”) strand.

The term “sample” as used herein is used in its broadest sense. Abiological sample suspected of containing nucleic acid can comprise, butis not limited to, genomic DNA (in solution or bound to a solid supportsuch as for Southern blot analysis), cDNA (in solution or bound to asolid support), and the like.

The term “urinary tract” as used herein refers to the organs and ductswhich participate in the secretion and elimination of urine from thebody.

The terms “transrenal DNA” and “transrenal nucleic acid” as used hereinrefer to nucleic acids that have crossed the kidney barrier.

The present invention encompasses a platform for the detection and genespecific analysis of transrenal DNA fragments carrying differentnucleotide lesions and adducts caused by various external and internalDNA modifying factors. Without limiting the scope of the invention, butin the interest of clarity, some factors that generate DNA modificationsmight be grouped in three classes: (i) physical, including but notlimited to gamma and UV irradiation, temperature fluctuations; (ii)chemical, including but not limited to environmental pollutants,naturally occurring genotoxins, carcinogens, anticancer drugs and (iii)reactive metabolites such as active forms of oxygen, lipid peroxidationproducts and hydrolytic agents.

I. APPLICATIONS OF THE METHODS OF THE PRESENT INVENTION

The present invention can be used for many applications, including,without in any way limiting the invention, the following.

A. Analyzing for the Presence of Fetal Nucleic Acids in Maternal Urine

The present invention provides methods of analyzing for the presence ofspecific fetal nucleic acid sequences or nucleic acid modifications bydetecting specific fetal nucleic acid sequences that have crossed theplacental and kidney barriers and are present in maternal urine. Themethods generally involve the steps of obtaining a urine sample from apregnant woman and subjecting the material to a method of detecting aspecific fetal nucleic acid sequence or modification of interest. In oneembodiment, the method further encompasses substantially purifyingnucleic acids present in the urine sample prior to detecting thespecific nucleic acid sequence or modification of interest. Thesemethods have a variety of diagnostic applications, including thedetermination of fetus sex and the identification of fetal geneticdiseases, such as those inherited from the father for various purposes,including determinations of paternity.

The inventions described herein can be used, for example, to diagnoseany of the more than 3000 genetic diseases currently known or to beidentified (e.g. hemophilias, thalassemias, Duchenne muscular dystrophy,Huntington's disease, Alzheimer's disease and cystic fibrosis). Anygenetic disease for which the mutation(s) or other modification(s) andthe surrounding nucleotide sequence is known can be identified bymethods of the present invention. Some diseases may be linked to knownvariations in methylation of nucleic acids, that can also be identifiedby methods of the present invention.

Further, there is growing evidence that some DNA sequences canpredispose an individual to any of a number of diseases such asdiabetes, arteriosclerosis, obesity, various autoimmune diseases andcancer (e.g. colorectal, breast, ovarian, lung), or chromosomalabnormality (either prenatally or postnatally). The diagnosis for agenetic disease, chromosomal aneuploidy or genetic predisposition can beperformed prenatally by collecting urine from the pregnant mother.

Urine DNA analysis provides an easier and safer way to perform prenataltesting. A fetus receives equal amount of genetic information from bothparents. The loss of a large number of fetal cells during development isa major part of the genetic program for embryonic differentiation andformation of a normal body. DNA from these dying embryonic cells notonly escapes into the bloodstream of the mother, but also crosses thekidney barriers where it appears in the mother's urine. As shown in thefollowing examples, pieces of the male-specific Y chromosome can befound in the urine of women pregnant with male fetuses. Example 8,below, shows that the fetal genetic information was found in themother's urine as early as the 7th to ₈ ^(th) week of pregnancy, thatis, at least 6-8 weeks earlier than can be obtained by eitheramniocentesis or chorionic villus sampling.

In one embodiment of this invention, a simple noninvasive test can beused for early determination of the fetal gender. However, there aremore far-reaching consequences of these findings with regard todevelopment of modern safe diagnostic techniques. The discovery that DNAfrom the developing embryo appears in the mother's urine presents theopportunity to quickly develop products for analysis of genes inheritedfrom the father. These include genes that contain disease-relatedmutations or can cause problems on different genetic backgrounds. As anexample, if a pregnant woman is Rh−(negative Rhesus factor) and producesanti-RhD antibodies and a father is Rh+, amniocentesis is currentlyrecommended for early diagnostics of Rh incompatibility, which oftencauses life threatening hemolytic anemia in the newborn baby. Detectionof the RhD gene-specific sequence in the mother's urine will be anexcellent alternative to amniocentesis, which is considered hazardous bya growing number of physicians worldwide. This test is also lessexpensive and more cost-effective, because it avoids the necessity of asurgical step in obtaining samples for analysis.

With the advent of broad-based genetic mapping initiatives such as theHuman Genome Project, there is an expanding list of targets andapplications for genetic analysis of urine DNA. Many diseases inheritedby the fetus will be easily detectable by analysis of the mother's urineDNA. These include Marfan Syndrome, Sickle Cell Anemia, Tay SachsDisease, and a group of neurodegenerative disorders, includingHuntington's Disease, Spinocerebellar Ataxia 1, Machado-Joseph Disease,Dentatorubraopallidoluysian Atrophy, and others that affect the fetusand newborn. Urine DNA analysis can detect the presence of the mutantgene inherited from the father. Also, if the mother's genome bears amutation, the test can help determine whether a normal version of thegene has been inherited from the father.

In addition to providing answers to commonly asked questions fromexpectant couples, determination of fetal sex can also be very helpfulif there is a risk of X chromosome-linked inherited disease, e.g.Hemophilia or Duchenne Muscular Dystrophy. Again prenatal testing forinherited diseases is currently performed with specimens obtained byamniocentesis. There are two major disadvantages of this technology:First, amniocentesis can only be performed after the 14th week ofpregnancy. Second, in some instances, the risk associated with aninherited disorder is comparable to the risk associated with thesurgical procedure of amniocentesis. Urine DNA based technology canpresent the information while avoiding both problems.

An important factor contributing to the success of any new diagnostictest is the necessity that patients and doctors express a preference forthe new test. Invasive prenatal testing is often declined by the patientbecause of the attendant risks to the fetus and mother. If the sameinformation can be obtained from a safe and simple urine test, it islikely that the test will be given widespread acceptance by the publicand medical community.

B. Analyzing for the Presence of Specific Host Nucleic Acid Sequencesthat Cross the Kidney Barrier

The present invention further provides methods enabling the detection ofspecific nucleic acid sequences originating from the patient's ownendogenous nucleic acid that must cross the kidney barrier to appear inthe urine. The method generally involves the steps of obtaining a urinesample from a patient and subjecting the material to a method ofdetecting a target nucleic acid sequence. In one embodiment, the methodfurther encompasses substantially purifying nucleic acids present in theurine sample prior to detecting the target nucleic acid. This method hasa variety of diagnostic applications, including, but not limited to,tumor diagnosis and the diagnosis of diseases caused by clonal expansionof cells containing DNA modifications accompanied by death of at least asubset of the cells bearing DNA modifications.

Success of tumor treatment is currently dependent on tumor type andmethod of treatment. However, the most important factor determining thesuccess of cancer therapy is detection of the tumor at the earliestpossible stage of development. The earlier a tumor is detected thebetter is the prognosis. In many pre-neoplastic conditions, such asinherited predisposition to a specific tumor type or a disease promotingneoplastic transformation, (e.g. chronic hepatitis and cirrhosis),significant efforts for early tumor detection are currently beingapplied but existing techniques are usually invasive and expensive. Theoncologist's arsenal now includes tests that are not only invasive,often hazardous, but also less reliable than expected.

From the patient's point of view, the invasive tests are expensive andsufficiently unpleasant to warrant decisions to forgo needed tests suchas rectocolonoscopy for diagnostics of colorectal cancer. The problem ofcompliance is of critical importance when high-risk patients areencouraged to submit to procedures that are clearly uncomfortable andunpleasant. Dramatic improvement of high-risk patient compliance is anabsolute necessity for the future. Thus, development of new methods forearly tumor detection is absolutely necessary for a substantial progressin this area of medicine. It is also clear that such methods should bebased not only on more sensitive techniques for detection of clinicalsymptoms of neoplastic growth, but rather on revealing tumorcell-specific markers.

The earliest cellular changes that can be used as a marker of neoplastictransformation are changes that cause the transformation, i.e. geneticand epigenetic DNA modification. Various changes in DNA sequences and/orin the methylation status of CpG islands (especially of those located inpromoter regions of tumor suppressor genes) are currently used as tumormarkers. As more such markers are discovered, it has become evident thatsome are characteristic of early tumor stages, or even of pre-neoplasticconditions. Other DNA modifications can indicate relatively late phasesof neoplastic transformation. Also there are expectations that somechanges in DNA sequences and its methylation pattern will help predictmetastatic potential and tumor cell sensitivity to differentchemotherapeutic agents. Cell death occurs at all stages of tumor growthand detection of tumor-specific changes in the urine DNA can be anexcellent marker for tumor diagnosis and monitoring of anti-tumortherapy. The example section below shows that tumor-specific mutationsof the K-ras gene can be detected in the urine of patients withcolorectal tumors that bear this mutation.

One of the greatest clinical challenges for tumor chemotherapy is thevariable sensitivity of different tumors to anti-tumor drugs, and theabsence of a simple test for the quick early stage evaluation ofanti-tumor therapy. Normally, the oncologist can observe the results oftreatment only after several months. Meanwhile, the tumor can continueto grow and possibly metastasize if the chemotherapeutic regimen isineffective. One embodiment of the present invention, useful for theimmediate monitoring of the effectiveness of tumor therapy, is thequantitative analysis of tumor-specific mutations in the patient's urineDNA. If the treatment is effective, then more tumor cells die, and theratio of the mutant sequence to any normal reference sequence containedin the urine will increase. Eventually, if chemotherapy is effective themutant tumor-specific sequence will disappear. Periodic analysis of apatient's urine DNA can be used for monitoring of possible tumorre-growth. Early indication of chemotherapeutic ineffectiveness wouldallow time to try other chemotherapeutics and anti-tumor treatments.This approach is similarly effective for the evaluation of theeffectiveness of radiation therapy and other cancer therapies and formonitoring after surgical treatment of cancerous growths.

C. Analyzing for the Presence of Specific Non-host Nucleic AcidSequences that Cross the Kidney Barrier

The present invention also provides methods enabling the detection ofspecific nucleic acid sequences that do not originate from the patient'sendogenous nucleic acid sequences, and must cross the kidney barrier toappear in the urine. The steps are the same as for the detection of hostoriginated nucleic acids, except that the detection method selects fornon-host nucleic acid sequences. This method has a variety of diagnosticapplications, including, but not limited to, diagnosis of infection bynucleic acid containing pathogens that infect areas other than theurinary tract, and do not shed nucleic acids directly into the urinarytract.

In one embodiment, the present invention has important applications inorgan and tissue transplantation science. Transplantation of differentorgans, tissues, and cells or other material that contains nucleic acids(referred to as “transplanted material”) is now widely used in clinicalpractice. The most important problem faced by the transplant patient andthe healthcare delivery system is the requirement to carefully controlthe normal immune response of the recipient that leads to transplantrejection and failure. In spite of intensive therapy designed tosuppress the recipient's immune response, rejection episodes often occurduring the post-transplantation period and their early detection can bevery useful, if not critical for effective clinical management.

Each person has a distinct and unique pattern of genes that are encodedby DNA. Since the donor's DNA is genetically different from therecipient's DNA, the present invention can be used to “monitortransplanted material” which is defined as detecting and/or measuringthe rejection or acceptance of transplanted organs, tissues and cells bythe recipient. This will reduce and even eliminate in some instances thenecessity of taking tissue biopsies from already debilitated patients.Example 3, below, describes a test for the appearance of Ychromosome-specific DNA sequences in the urine of female recipients whohad received blood transfusions with blood from males. These experimentsshowed that due to the death of white blood cells from the male donor, Ychromosome-specific sequences appeared in the urine of the femalerecipient. These blood cells die in much the same manner as the cells ofa transplanted organ that has been attacked by the recipient's immunesystem. Methods of the present invention can be used to track theprogress of recipients of cell, tissue and organ transplants.

D. Analyzing the Form and Degree of Methylation of the Target DNA

Changes in DNA methylation of specific genomic areas affect chromatinstructure and DNA transcription, and consequently, are beinginvestigated for their involvement in various pathological processes.Analysis of transrenal DNA methylation would be a useful diagnostictool.

Mutations and changes in DNA methylation status that happen during tumorprogression can be used as the tumor markers. Esteller M, et al.,Detection of Aberrant Promoter Hypermethylation of Tumor SuppressorGenes in Serum DNA from Non-Small Cell Lung Cancer Patients, Cancer Res1999; 59: 67-70. Wong IH, et al., Detection of aberrant p 16 methylationin the plasma and serum of liver cancer patients, Cancer Res 1999; 59:71-3. Various changes in DNA sequences and/or in the methylation status(Baylin SB, et al., Alterations in DNA methylation: A fundamental aspectof neoplasia, Adv Canc Res 1998; 72:141-96) of CpG islands (especiallyof those located in promoter regions of tumor suppressor genes) arecurrently used as tumor markers. Also there are expectations that somechanges in DNA sequences and its methylation pattern will help topredict metastatic potential and tumor cell sensitivity to differentchemotherapeutic agents. methylation in CpG islands of some genes, e.g.MYF-3 gene, can be bound to different stages of carcinogenesis.Hypermethylation of this gene in comparison with normla mucosa wasobserved in 88% of adenomas and 99% of carcinomas. Shannon B, et.al.,Hypermethylation of the MYF-3 gene in colorectal cancers: associationswith pathological features and with microsatellite instability, Int JCancer 1999; 84:109-13.

There are no reliable markers based on DNA mutations for HCC. However,in this case there is a growing group of markers that are based on CpGisland methylation in a gene promoter region, e.g. the p16 or GSTPIpromoter. p16 methylation was found in more than 70% of HCC tissues andamong HCC cases with aberrant methylation similar changes were alsodetected in about 80% of the plasma samples. Wong IH, et al., Detectionof aberrant p16 methylation in the plasma and serum of liver cancerpatients, Cancer Res 1999; 59:71-3. Matsuda Y, et al., p16(INK4) isinactivated by extensive CpG methylation in human hepatocellularcarcinoma, Gastroenterology 1999; 116:394-400. Somatic hypermethylationof GSTP1 CpG islands was observed in DNA from more than 80% of HCCcases. Tchou JC, et al., GSTP1 CpG island DNA hypermethylation inhepatocellular carcinomas, Int J Oncol 2000; 16:663-76.

We have already discussed how methylation of CpG islands in promoters oftumor suppressor genes leading to their inactivation, is involved inpre-neoplastic conditions and carcinogenesis, and can be used fordiagnostics of those pathological processes. Methylation ofestrogen-receptor gene has been linked to heart disease (Fricker J.Heart disease linked to oestrogen-receptor gene methylation. Mol. Med.Today, 5, 505-506, 1999). Fragile X chromosome syndrome is associatednot only with the expansion of the number of CGG trinucleotide tandemrepeats at the 5′ untranslated region of the FMR1 gene but also withhypermethylation in the CGG repeats and the adjacent CpG islands(Panagopoulos I, et al., A methylation PCR approach for detection offragile X syndrome. Hum. Mutat., 14, 71-79, 1999). Analysis of themethylation status at the CpG islands of the small nuclearribonucleoprotein associated polypeptide N (SNRPN) gene using amnioticfluid cell cultures or cultivated chorionic villus samples has beenrecommended for prenatal diagnosis of Prader-Willi and Angelmansyndromes (Kubota T, et al., Analysis of parent of origin specific DNAmethylation at SNRPN and PW71 in tissues: implication for prenataldiagnosis. J. Med. Genet., 33, 1011-1014, 1996). DNA hypermethylation ofthe promoter region of the E-cadherin gene is characteristic of chronichepatitis and liver cirrhosis (Kanai Y, et al., Aberrant DNA methylationprecedes loss of heterozygosity on chromosome 16 in chronic hepatitisand liver cirrhosis. Cancer Lett., 148, 73-80, 2000). Of course manymore modifications in DNA methylation status will be linked to variousdiseases in the future. Detection of those modifications in transrenalDNA will be a useful marker in prenatal testing as well as for diagnosisof pathological processes in adult organisms.

It is known that aging is accompanied by specific changes in the genomemethylation status, hypermethylation of some CpG islands anddemethylation in coding regions of genome (Toyota M and Issa J-PJ. CpGislands methylator phenotypes in aging and cancer, Seminars in CancerBiol., 9, 349-357, 1999). Detection of these changes in transrenal DNA,that contains DNA fragments from various cell types, can be used as amarker of normal and pathological aging processes.

II. METHODS FOR NUCLEIC ACID MANIPULATION AND DETECTION

Techniques for nucleic acid manipulation useful for the practice of thepresent invention are described in a variety of references, includingbut not limited to, Molecular Cloning: A Laboratory Manual, 2nd ed.,Vol. 1-3, eds. Sambrook et al. Cold Spring Harbor Laboratory Press(1989); and Current Protocols in Molecular Biology, eds. Ausubel et al.,Greene Publishing and Wiley-Interscience: New York (1987) and periodicupdates. Specific descriptions, while not intended to limit the scope ofthe present invention, provide guidance in practicing certain aspects ofthe present invention.

A. Reducing Degradation by DNase

DNA is subject to degradation by DNases present in urine. The presentinvention encompasses several methods for preventing or reducing thedegradation of DNA while in urine so that sufficiently large sequencesare available for detection by known methods of DNA detection such asthose described below. In one embodiment, samples of urine are takenwhen the urine has been held in the bladder for less than 12 hours, in aspecific embodiment the urine is held in the bladder for less than 5hours, more preferable for less than 2 hours. Collecting and analyzing aurine sample before it has been held in the bladder for a long period oftime reduces the exposure of DNA to the any DNase present in the urine.

In another embodiment of the present invention, after collection, theurine sample is treated using one or more methods of inhibiting DNaseactivity. Methods of inhibiting DNase activity include, but are notlimited to, the use of ethylenediaminetetraacetic acid (EDTA),guanidine-HCI, GITC (Guanidine isothiocyanate), N-lauroylsarcosine,Na-dodecylsulphate (SDS), high salt concentration and heat inactivationof DNase.

In yet another embodiment, after collection, the urine sample is treatedwith an adsorbent that traps DNA, after which the adsorbent is removedfrom the sample, rinsed and treated to release the trapped DNA fordetection and analysis. This method not only isolates DNA from the urinesample, but, when used with some adsorbents, including, but not limitedto Hybond N membranes (Amersham Pharmacia Biotech Ltd., Piscataway,N.J.) protects the DNA from degradation by DNase activity.

B. Increasing Sensitivity to Detection

In some cases, the amount of DNA in a urine sample is limited. Therefor,for certain applications, the present invention encompasses embodimentswherein sensitivity of detection is increased by any method(s) known inthe art, including, without limitation, one or more of the followingmethods.

Where DNA is present in minute amounts in the urine, larger urinesamples can be collected and thereafter concentrated by any means thatdoes not effect the detection of DNA present in the sample. Someexamples include, without limiting the breadth of the invention,reducing liquid present in the sample by butanol concentration orconcentration using Sephadex G-25 (Pharmacia Biotech, Inc., PiscatawayN.J.).

Nested PCR can be used to improve sensitivity by several orders ofmagnitude. Because of the vulnerability of nested PCR to inaccurateresults due to DNA contamination, in one embodiment of the presentinvention, precautions are taken to avoid DNA contamination of thesample. For example, without limiting the present invention, one cantreat PCR reagents with restriction endonuclease(s) that cleave withinthe target sequence, prior to adding them to the test DNA sample.

C. Substantially Purifying Nucleic Acids Prior to Detection

In one embodiment, the present invention encompasses substantiallypurifying or isolating nucleic acids from a sample prior to detection.Nucleic acid molecules can be isolated from urine using any of a numberof procedures, which are well-known in the art. Any method for isolationthat facilitates the detection of target nucleic acid is acceptable. Forexample, DNA can be isolated by precipitation, as described by Ishizawaet al., Nucleic Acids Res. 19, 5972 (1991). Where a large volume samplecontains a low concentration of DNA, as with urine, a preferred methodof isolating DNA is encompassed. In this method, a sample is treatedwith an adsorbent that acts to concentrate the DNA. For example, asample can be treated with a solid material that will adsorb DNA, suchas, without limitation, DEAE Sephadex A-25 (Pharmacia Biotech, Inc.,Piscataway N.J.), a DNA filter, and/or glass milk. Sample DNA is elutedfrom the adsorbent after other compositions are washed away.

In consideration of the sensitivity of various nucleic acid analyzingtechniques, such as PCR, the present invention also encompasses methodsof reducing the presence of contaminating nucleic acids in the urinesample. Contamination of urine samples by nucleic acid sequences thathave not crossed the kidney barrier can be introduced by cells sheddingfrom the urinary tract lining, by sexual intercourse, or duringprocessing of the urine sample prior to detection of the DNA sequence ofinterest. Without intending to limit the present invention to anymechanism, it is believed that DNA passing the kidney barrier andappearing in urine is likely to have on average a shorter length thanDNA introduced from contaminating sources because of the fragmentationthat occurs in apoptotic cells and necrotic cells in the body, combinedwith the action of DNase in the blood and urine.

Filtration can be used to reduce the level of contaminating DNA in aurine sample prior to detection, by selecting for shorter sequences ofDNA. In one embodiment of the present invention nucleic acids containingmore than about 1000 base pairs, or 1000 nucleotides when denatured, areremoved from the sample prior to detection. In a specific embodiment ofthe present invention, urine samples are filtered prior to amplificationby PCR to remove substantially all DNA comprising greater than 300 basepairs, or 300 nucleotides when denatured. Without limiting the inventionto a specific mechanism, it is proposed that such a filtration removescontaminating DNA from cells shed from the urethral/bladder wall orintroduced into the urethra during sexual intercourse. The majority ofDNA from such contaminating sources are likely to comprise more than 300nucleotides as the DNA is not for the most part a product offragmentation of nucleic acids as a result of apoptotic cell death.

Nucleic acid molecules can also be isolated by gel electrophoresis,whereby fragments of nucleic acid are separated according to molecularweight. The technique of restriction fragments length polymorphisms(RFLP), applies the methods of electrophoresis separation, followed bynucleic acid detection enabling comparison by molecular weight offragments from two or more alleles of a specific gene sequence.

The above-mentioned methods of purification are meant to describe, butnot limit, the methods suitable for use in the invention. The methods ofisolating nucleic acids are within the ability of one skilled in the artand are not described in detail here.

D. Analysis and Detection of Specific Nucleic Acid Sequences

The expression “assaying for the presence of a nucleic acid sequence”refers to the use of any method to determine whether or not a nucleicacid sequence is present in a sample. Methods include, but are notlimited to, techniques for hybridization, amplification and detection ofnucleic acids. One skilled in the art has access to a multitude of thesemethods, including, but not limited to, those set forth in CurrentProtocols in Molecular Biology, eds. Ausubel et al., Greene Publishingand Wiley-Interscience: New York (1987) and periodic updates. It iscontemplated that two or more methods can be used in combination toconfirm the results or improve the sensitivity of the assay. An exampleof analyzing by the combination of methods to determine whether or not anucleic acid sequence is present is the technique of restrictionfragment length polymorphism based PCR (“PCR-RFLP”), where nucleic acidsequences are amplified, treated with restriction enzymes, and separatedby electrophoresis, allowing for the detection of nucleic acidscontaining small modifications, such as point mutations.

The terms “detect” and “analyze” in relation to a nucleic acid sequence,refer to the use of any method of observing, ascertaining or quantifyingsignals indicating the presence of the target nucleic acid sequence in asample or the absolute or relative quantity of that target nucleic acidsequence in a sample. Methods can be combined with nucleic acid labelingmethods to provide a signal by, for example: fluorescence,radioactivity, colorimetry, gravimetry, X-ray diffraction or adsorption,magnetism, enzymatic activity and the like. The signal can then bedetected and/or quantified, by methods appropriate to the type ofsignal, to determine the presence or absence, of the specific DNAsequence of interest.

To “quantify” in relation to a nucleic acid sequence, refers to the useof any method to study the amount of a particular nucleic acid sequence,including, without limitation, methods to determine the number of copiesof a nucleic acid sequence or to determine the change in quantity ofcopies of the nucleic acid sequence over time, or to determine therelative concentration of a sequence when compared to another sequence.

To assist in detection and analysis, specific DNA sequences can be“amplified” in a number of ways, including, but not limited to cyclingprobe reaction (Bekkaoui, F. et al, BioTechniques 20,240-248 (1996),polymerase chain reaction (PCR), nested PCR, PCR-SSCP (single strandconformation polymorphism), ligase chain reaction (LCR) (F. Barany Proc.Natl. Acad. Sci USA 88:189-93 (1991)), cloning, strand displacementamplification (SDA) (G. K. Terrance Walker et al., Nucleic Acids Res.,22:2670-77 (1994), and variations such as allele-specific amplification(ASA).

An alternative to amplification of a specific DNA sequence that can beused to indicate the presence of that sequence in methods of the presentinvention is based on hybridization of a nucleic acid cleavage structurewith the specific sequence, followed by cleavage of the cleavagestructure in a site-specific manner. This method is herein referred toas “cleavage product detection.” This method is described in detail inU.S. Pat. Nos. 5,541,331 and 5,614,402, and PCT publication Nos. WO94/29482 and WO 97/27214. It allows for the detection of small amountsof specific nucleic acid sequences without amplifying the DNA sequenceof interest.

E. Detection, Analysis and Quantification of Methylated Regions of DNA

Without limiting the present invention to any specific methods ofdetection, analysis or quantification of methylated regions of DNA, thefollowing techniques are useful for evaluating DNA methylation. Methodsfor the mapping and quantification of methylated regions of DNA, ingeneral, and for analysis of transrenal DNA, in particular, may begrouped in two classes: methods allowing to assess overall methylationstatus of CpG islands and methods for analysis of sequence specificmethylation.

Methods in the first group rely on Southern hybridization approach,based on utilization of properties of methylation sensitive restrictionnucleases. Hatada I, et al., Proc Natl Acad Sci USA 1991 Nov 1, 199188(21):9523-7, describes a genomic scanning method for higher organismsusing restriction sites as landmarks. Issa JP, et al., Nat Genet Aug. 7,1994. (4):536-40, shows that methylation of the oestrogen receptor CpGisland links ageing and neoplasia in human colon. Pogribny I. and Yi P,James SJ, Biochem Biophys Res Commun Sep. 7, 1999 262(3):624-8, describea sensitive new method for rapid detection of abnormal methylationpatterns in global DNA with and within CpG islands.

Recently designed DNA microarray based technology can also be includedin this group. Huang TH, et al., Genet Mar. 8, 1999(3):459-70, describesmethylation profiling of CpG islands in human breast cancer cells.

The methods in the second group are based on registration of thesequence differences between methylated and unmethylated allelesresulting from bisulfite treatment of DNA. Registration usually iscarried out by PCR amplification using primers specific to methylatedand unmethylated DNA. Herman JG, et al., Proc Natl Acad Sci USA Sep. 3,1996; 93(18):9821-6, describes methylation-specific PCR: a novel PCRassay for methylation status of CpG islands. Depending on theexperimental setting several approaches based on this strategy have beendeveloped.

There are also several options for the quantification of methylated CpGislands in small amount of DNA (Xiong Z. and Laird PW, Nucleic Acids ResJun. 15, 1997; 25(12):2532-4, describing COBRA: a sensitive andquantitative DNA methylation assay, and Olek A, et al., Nucleic AcidsRes Dec. 15, 1996; 24(24):5064-6, describing a modified and improvedmethod for bisulphite based cytosine methylation analysis) and partiallydegraded DNA received from micro-dissected pathology sections (GonzalgoML and Jones PA, Nucleic Acids Res Jun. 15, 1997; 25(12):2529-31,describing rapid quantitation of methylation differences at specificsites using methylation-sensitive single nucleotide primer extension(Ms-SNuPE)). Additionally, there is a methylation sensitive SSCP thatwas developed for the analysis multiple methylation sites in CpG islands(Kinoshita H, et al., Anal Biochem Feb. 15, 2000; 278(2):165-9,describing methods for screening hypermethylated regions bymethylation-sensitive single-strand conformational polymorphism) and anextremely sensitive methylation specific Real Time PCR (Eads CA, et al.,Nucleic Acids Res Apr. 15, 2000; 28(8):E32, describing MethyLight: ahigh-throughput assay to measure DNA methylation).

F. Detection, Analysis and Quantification of Some Other Nucleic AcidModifications

Ligation-mediated polymerase chain reaction (LMPCR) has been used forthe detection of DNA adducts at individual nucleotide positions inmammalian genes. Adduct-specific enzymes, such as T4 endonuclease V,base excision repair enzymes, like UvrABC nuclease, and chemicalcleavage can be used to convert the adducts into DNA strand breaks. Thepositions of these breaks are then detected by LMPCR. Yoon JH, and LeeCS, Mol Cells Feb. 29, 2000; 10(1):71-5, describes the mapping ofaltromycin B-DNA adduct at nucleotide resolution in the human genomicDNA by ligation-mediated PCR. Pfeifer GP, et al., Proc Natl Acad Sci USAFeb. 15, 1991; 88(4):1374-8, describes the in vivo mapping of a DNAadduct at nucleotide resolution: detection of pyrimidine (6-4)pyrimidone photoproducts by ligation-mediated polymerase chain reaction.Pfeifer GP, Denissenko MF, and Tang MS, Toxicol Lett Dec. 28, 1998;102-103:447-51, describes PCR-based approaches to adduct analysis.

Using this approach the distribution of benzo[a]pyrene diol epoxideadducts (formed by cigarette smoke major carcinogen benzo[a]pyrene) inthe P53 gene was mapped at nucleotide resolution. Adduct formation wasobserved at the nucleotide positions that appeared to be mutationalhotspots in human lung cancers. Denissenko MF, et al., Science Oct. 18,1996; 274(5286):430-2, describes preferential formation ofbenzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Asimilar trend was observed in the case of skin cancer. Tommasi S,Denissenko MF, and Pfeifer GP, Cancer Res Nov 1, 1997; 57(21):4727-30,shows that sunlight induces pyrimidine dimers occur preferentially at5-methylcytosine bases. Thus, the distribution of DNA adducts in the p53gene caused by environmental carcinogens corresponds to the mutationalhotspots of certain cancers.

These data indicate that both quantitation of DNA adducts and their genespecific nucleotide mapping in transrenal DNA can be used for theevaluation of genotoxic effects of environmental factors, dietary andother carcinogens as well as for prediction of resulting predispositionto a specific type of cancer.

The following examples are provided to illustrate but not limit theinvention

EXAMPLE 1 DETECTION OF POLYMERIC DNA IN URINE OF MICE PREINOCULATED WITHλ PHAGE DNA

This example analyzes the ability of DNA to cross the kidney barrier inrodents and appear in detectable form in urine.

λ phage DNA (New England BioLabs, MA) was labeled by nick translationwith [α-³²P] dNTP DNA (New England BioLabs, MA) using the Klenowfragment of E. Coli DNA polymerase to a specific radioactivity of 10⁸cpm/μg as previously described. (/]Sambrook J., Fritsch E. F., ManiatisT., Molecular Cloning. A Laboratory Manual. 2d Edition. Cold SpringHarbor Laboratory Press, 1989). Two months old male Wistar rats wereinjected subcutaneously with 0.4 μg of the [³²p] labeled DNA. Urinesamples were then collected for three days and total and acid-insolubleradioactivity was measured in a liquid scintillation counter. Thekinetics of excretion of acid-insoluble radioactivity in urine appear inTable 1, below. It was determined that 3.2% of the total DNA with whichthe rats were inoculated crossed the kidney barrier and was detected inurine and 0.06% of the total DNA appeared in the urine in anacid-insoluble form; representing polymeric nucleotides.

TABLE 1 KINETICS OF URINE EXCRETION OF INJECTED [³²P]DNA 1st day 2nd day3rd day TOTAL TOTAL RADIOACTIVITY (CPM) 1,080,800 100,800 7,7001,189,300 (% INJECTED DNA) (2.9%) (0.3%) (0.02%) (3.2%) ACID-INSOLUBLERADIOACTIVITY(CPM)  21,000 ND ND  21,000 (% INJECTED DNA)  (0.06%) (0.06%)

DNA from the urine samples was isolated by phenol deproteinization(Sambrook J., Fritsch E. F., Maniatis T. Molecular Cloning. A LaboratoryManual. 2d Edition. Cold Spring Harbor Laboratory Press, 1989) andsubjected to electrophoresis in a 1.5% agarose gel. Gels were stainedwith ethidium bromide (0.5 μg/ml) for visualization of DNA. (SambrookJ., Fritsch E. F., Maniatis T. Molecular Cloning. A Laboratory Manual.2d Edition. Cold Spring Harbor Laboratory Press, 1989).

FIGS. 1A and 1B depict the results of duplicate experiments (representedby lanes 1 and 2). FIG. 1A is the gel viewed by ethidium bromidestaining, and FIG. 1B represents the same gel viewed by autoradiography.Labeled fragments of DNA appeared in the autoradiography representingsequences averaging approximately 150 base pairs.

The results of this experiment support that DNA can both cross thekidney barrier in polymeric form and remain in polymeric form in urine,notwithstanding the presence of DNases, for a period of time sufficientto allow a urine sample to be taken and DNA to be isolated from theurine sample. The kinetics of excretion of the injected DNA suggest thatmuch of the injected DNA is reutilized in the body prior to appearing inthe urine. It is possible that recycled radiolabeled nucleotides couldlater be incorporated into cells from the urinary tract and then appearin the urine without crossing the kidney barrier. However, it isunlikely that the DNA detected in this experiment could have beenintroduced into the urine from cells of the urinary tract, due to theshort period of time between injection of labeled DNA and urinecollection. While the injected DNA may eventually appear in cells of thebladder, it is unlikely to represent the same nucleic acid sequencebecause it is expected that the action of nucleases in the body willafter a sufficient period of time cell free in the body, eventuallybreak down the DNA to nucleotides.

EXAMPLE 2 DETECTION OF HUMAN DNA SEQUENCES IN URINE OF MOUSEPREINOCULATED WITH HUMAN CELLS

Example 1 showed that DNA sequences could remain in polymeric form inthe blood stream, cross the kidney barrier, and remain suitable forsubsequent detection. The next set of experiments were performed todetermine if DNA from cells dying in the organism but not in the urinarytract can be detected in urine.

Human Raji lymphoma cells growing in RPMI supplemented with 5% fetalcalf serum were irradiated with 1000 rads of ¹³⁷CS γ-rays. Mice werethen inoculated subcutaneously with 10⁸ cells each. Urine samples werecollected for three days, and DNA was isolated as described above.Human-specific DNA sequences were detected by multilocus screening usingAlu oligonucleotide-directed PCR as previously described, (Zietkiewicz,E., Labuda M., Sinnett D., Glorieux F. H., and Labuda D., Proc. Natl.Acad. Sci. USA, 89, 8448-8451, 1992), followed by electrophoresis in a1.5% agarose gel.

The results appear in FIG. 2. PCR amplification of urine DNA from thecontrol animal (lane 1) did not produce any DNA fragments; and thefragments obtained from PCR amplification of the urine DNA taken fromthe test mouse that was injected with human cells (lane 3) did containdetectable human DNA sequences, as evidenced by a comparison with theidentical bands that appear in the reference human DNA sample (lane 2).

The results support that a portion of DNA from cells dying in a mammalcrosses the kidney barrier and remains in polymeric form in urine,notwithstanding the presence of DNases, for a period of time sufficientto allow a urine sample to be taken and DNA to be isolated from theurine sample. Further, one can test for the presence of specific DNAsequences in urine samples using methods such as PCR amplification ofspecific desired sequences that would not be present in the urine samplebut for having crossed the kidney barrier in amplifiable form.

EXAMPLE 3 DETECTION OF TRANSRENAL NUCLEIC ACIDS

Taken together, Examples 1 and 2 demonstrate that, in the mouse model,both free DNA and DNA from dying cells cross the kidney barrier and canbe detected in urine by PCR analysis. Two systems were selected asmodels to demonstrate that DNA can cross the kidney barrier and remainin polymeric form in human urine samples. The systems are designed tofocus on DNA originating from cells dying outside the urinary tractrather than DNA that appears in urine due to the death of cells in theurinary tract.

Women who were either pregnant or transfused with male blood werestudied because both of these models represented humans having DNA intheir bodies that is not present in their normal genome. Each womanstudied was analyzed for the presence of Y chromosome-specific sequencesin their urine.

Detection of repeated and Single Copy Y Chromosome Specific Sequences inthe Urine of Women Pregnant with Male Fetuses.

As discussed above, apoptotic cell death plays a significant role inembryogenesis. If fetal DNA crosses the placental barrier, it willappear in the mother's blood and, subsequently, in her urine. Fetalreticulocytes and white blood cells are other potential sources of fetalDNA and are also detected in the mother's blood in the first 4-5 weeksof pregnancy. Lo, Y-M. D., et al., Lancet 335:1463-1464, (1990).Bianchi, D. W., et al., Proc. Natl. Acad Sci. USA 87:3279-3283, (1990).Bianchi, D. W., et al., Proc. Natl. Acad. Sci. USA 93:705-708, (1996).

Urine samples were obtained from pregnant women at gestational ages ofbetween 16 and 36 weeks. Fetal sex was confirmed by ultrasoundscreening. In previous studies, described in Example 4, below, it wasfound that human urine contains components that can degrade DNA (DNase).This DNase activity varies from sample to sample. To reduce DNAdegradation, the following steps were taken to collect and preserve theurine samples. Urine samples (20 ml each) were collected in 50 ml Comingtubes filled with 5 ml of 250 mM ethylenediaminetetraacetic acid (EDTA)solution as a prophylactic against DNase activity. Tubes containing theurine samples were then kept at −80° C. until use.

Two methods were used for DNA isolation from the urine samples, both ofwhich gave essentially the same results. In the first method, previouslydescribed by Ishizawa et al. (Ishizawa M., et al., Nucleic Acids Res.19, 5972, 1991), urine samples (150 μl) were thawed at 60° C. and addedto 3 volumes of a solution containing 6 M guanidine isothyocyanate(GITC), 13 mM EDTA, 0.5% sodium N-lauroylsarcosine, 10 μg glycogen, and26 mM Tris-HCl, pH 8. The mixture was incubated at 60° C. for 15 min. ina heating block and DNA was precipitated by addition of an equal volumeof isopropanol. After vigorous shaking tightly capped tubes were keptfor 15 min. at room temperature and DNA was collected by centrifugationat 10,000×g for 5 minutes. The resultant pellet was washed with 80%ethanol, air-dried, dissolved in 50 μl of deionized water and treatedwith Chelex 100-based DNA extraction reagent (Perkin Elmer) as described(Walsh P. S., Metzger D. A., Higuchi R. BioTechniques 10, 506-513,1991).

The present invention encompasses an alternative method of DNA isolationthat is suitable for the isolation of DNA from larger samples. In thismethod, based on DNA adsorption on glass powder, 2.5 ml urine sampleswere added to an equal volume of 10 M guanidine-HCl, and the mixture wasapplied to a column with 0.1 ml of 5% Wizard resin (Wizard Minipreps DNApurification system, Promega). The columns were washed with a solutioncontaining 100 mM NaCl in a 50% ethanol solution and the DNA was elutedwith 100 μl of deionized water.

DNA samples purified from urine were heat denatured for 5 minutes at 95°C. followed by filtration through a Microcon 100 filter (Amicon, MA) asrecommended by the supplier to separate from the sample substantiallyall DNA greater than 300 nucleotides in length.

Next, the samples were subjected to PCR. Each experiment had internalpositive and negative controls as well as a blank sample (salinesolution which was processed in the same way as the urine samples)designed to detect PCR contamination.

To reduce the chance of carryover DNA contamination of the PCR reactionmixture, the reagents were decontaminated prior to addition of a DNAsample by incubation with a restriction endonuclease specific for thetarget sequence: HinfI - for the Y chromosome-specific 97 base pairsequence and HaeIII for the 154 base pair sequence. The reagents weretreated for 1 hour at 37° C. with I unit per 25 μl of reaction mixture.PCR samples were then placed into a thermocycler cell, heated at 94° C.for 3 min. to inactivate the enzyme and DNA samples were added.

Two different markers were used to detect Y chromosome-specificsequences. DYZ1 is a repeated (2000-5000 times per genome) sequencedescribed by Nakahori Y., et al., Nucl. Acids Res. 14, 7569-7580, 1986.The single-copy gene marker DYS14 was also used to examine the abilityof the methods of the present invention to detect modifications insingle copy genes, such as occurs in certain genetic disorders andcancers. (Arnemann J., et al., Nucl. Acids Res. 15, 8713-8724, 1987).The single-copy sequence was analyzed using nested PCR, a more sensitiveand specific PCR technique. (Lo Y-M. D., et al., Lancet 335, 1463-1464,1990).

The following primers were used to amplify DYZ1 fragments:

(Y 1) 5′-TCCACTTTATTCCAGGCCTGTCC (SEQ ID NO: 1)

(Y2) 5′-TTGAATGGAATGGGAACGAATGG (SEQ ID NO: 2)

(YZ1) 5′-CCATTCCTTTGCATTCCGTTTCC (SEQ ID NO: 3)

(YZ2) 5′-ATCGACTGGCAGGGAACCAAAAG (SEQ ID NO: 4)

To detect DYS14 we performed nested PCR using the following primers:

(Y1.5) 5′-CTAGACCGCAGAGGCGCCAT (SEQ ID NO: 5)

(Y1.6) 5′-TAGTACCCACGCCTGCTCCGG (SEQ ID NO: 6)

(Y1.7) 5′-CATCCAGAGCGTCCCTGGCTT (SEQ ID NO: 7)

(Y1.8) 5′-CTTTCCACAGCCACATTTGTC (SEQ ID NO: 8)

Y1 and Y2 result in a 154 base pair product. Ivinson A. J., Taylor G.R.In PCR. A practical approach. (McPherson M. J., Quirke P., and Taylor G.R., eds.). IRL Press. Oxford, New York, Tokyo, pp. 15-27, 1993.

Shorter segments of DNA are believed to be more prevalent in the urinesamples than are longer segments due to filtration by the kidney barrierand the action of DNase. The present invention encompasses the novelprimers YZ1 and YZ2, that generate a shorter (97 base pairs) fragment,in order to maximize the power of the detection method.

To detect DYS14 we used nested PCR with the following primers: Y1.5 andY1.6 producing a 239 base pair external fragment; and Y1.7 and Y1.8producing a 198 base pair internal fragment. (Lo Y-M. D., et al., Lancet335, 1463-1464, 1990).

Thirty five or forty cycles of PCR reaction were performed. Cycleconditions were as follows: denaturation at 94° C. for 30 seconds;annealing at 58° C-63° C. (depending on primers, as described below) for60 seconds; chain elongation at 72° C. for 30 seconds. The denaturationstep was extended to 2 minutes at the beginning of the first cycle andthe last chain elongation step was extended to 7 minutes. Annealing wasat 63° C. for the YZ1/YZ2 primers and 58° C. for the Y1/Y2 primers. Fornested PCR forty cycles with the Y1.5/Y1.6 primers were followed by 25cycles with the Y1.7/Y1.8 primers, both at 58° C.

PCR products were analyzed in a 10% polyacrylamide gel (29: 1), 1×TBEelectrophoresis buffer, 10 V/cm, for 2.5 hours at room temperature andvisualized by ethidium bromide staining.

The results appear in FIGS. 4A, 4B and 5. In FIG. 4A, a 154 base pairPCR product of DYZ1, a repeated sequence of the Y chromosome, wasdetected with Y1 and Y2 primers. Lane M is an msp1 digest of pBR322 as amolecular weight standard; The negative control (lane 1, no DNA added)showed no detectable bands; the positive controls (lanes 2-5,representing 0. 1, 1.0, 10 and 100 pg of male total DNA, respectively)display bands of increasing intensity; the first group of test samples,from women carrying male fetuses (lanes 6 and 8), display clear bands ofthe same size as those in the positive controls; the second test sample,from a woman carrying a female fetus (Lane 7) displays no bands; twomore control lanes (9 representing a blank sample and 10 representingDNA from the urine of a non-pregnant woman) show no evidence of bands.

In FIG. 4B, a 97 base pair PCR product of DYZ1 was detected with YZ-1and YZ-2 primers. Lane M is an msp1 digest of pBR322 as a molecularweight standard; the positive controls (lanes 1-3, representing 0.1,1.0, and 10 pg of male total DNA, respectively) display bands ofincreasing intensity; the first test samples, from women carrying femalefetuses (lanes 4 and 5) displayed no bands; the second group of testsamples, from women carrying male fetuses (lanes 6 and 7), display bandsof the same size as those in the positive controls; two more controllanes (8 representing a blank sample and 9 representing DNA from theurine of a non-pregnant woman) show no evidence of bands.

In FIG. 5, a Y chromosome-specific single-copy DNA sequence (198 basepairs) was detected in the urine of pregnant women carrying malefetuses. Lane M is an msp1 digest of pBR322 as a molecular weightstandard; The negative control (lane 1, no DNA added) showed nodetectable bands; the positive controls (lanes 2-5, representing 0. 1,1.0, 10 and 100 pg of male total DNA, respectively) display bands ofincreasing intensity; the first group of test samples, from womencarrying male fetuses (lanes 6 and 7), display clear bands of the samesize as those in the positive controls; the second test sample, from awoman carrying a female fetus (Lane 8) displays no bands; two morecontrol lanes (9 representing a blank sample and 10 representing DNAfrom the urine of a non-pregnant woman) show no evidence of bands.

The results of these experiments support the following conclusionsregarding the present invention: a fraction of DNA from cells dying inthe animal or human body crosses the kidney barrier and can be detectedin urine in polymeric form, notwithstanding the presence of DNases; afraction of DNA from cells dying in the developing embryo crosses boththe placental and kidney barriers and can be detected in mother's urine;the size of the cell-free urine DNA is sufficient to be amplified inPCR; and the concentration of fetal DNA in mother's urine, even in thefirst few months of pregnancy, is high enough to detect genes whichexist only in single copy form in the fetal genome.

The results further support that maternal urine can be used as anindicator of fetal sex in specific and the composition of the fetalgenome in general, where it differs from the maternal genome, which canbe used for diagnosis of existing or potential disease. Analysis offetal DNA in a pregnant mother's urine can be used for detection ofsequences of DNA inherited from the male parent, including thosesequences indicative of or causing disease. Thus, the results supportthat methods of the present invention encompass the determination offetal sex as well as the diagnosis of certain fetal conditions which arecharacterized by the presence of specific DNA sequences in the fetalgenome.

Detection of Y Chromosome Specific Sequences in the Urine of a Woman whohas been Transfused with Male Blood.

In the case of a female transfused with a male donor's blood, thedonor's dead or dying white blood cells were expected to be the sourceof sequences of DNA specific to the male genome in the cell-free DNA ofthe recipient's blood and urine.

A urine sample was obtained from a woman 10 days following a transfusionwith 250 ml whole blood from a male donor. DNA from the urine wasisolated and tested for the presence of a male-specific 154 base pairsequence from the Y chromosome, using Y1 and Y2 primers, by using themethods described above.

The results appear in FIG. 3. Lane 1 is an Msp1 digest of pBR322 as amolecular weight standard; Lane 2 is a positive control (0.1 μg of totalDNA from lymphocytes of a male donor) displaying a 154 base pair band ofDNA; Lane 3 is a blank sample (saline solution passed through all theprocedures of DNA isolation and analysis); Lane 4 is a negative control(contains no added DNA); and Lane 5, displaying a 154 base pair sequencespecific to the Y chromosome, is DNA from the woman's urine samplefollowing blood transfusion. In a subsequent study, of nine womentransfused with male donor blood, a male specific band was detected infive samples. (Data not shown). Thus, in an embodiment of the presentinvention, various methods discussed herein, as well as any methodsknown in the art, can be used to improve the sensitivity of the method.

The results obtained show that, in a human, polymeric DNA released fromdying blood cells can remain in polymeric form in circulating blood,cross the kidney barrier and be detected in urine by PCR. The resultsfurther show that DNA sequences released from cells with genotypesdifferent from patients normal genotype can be selectively detected inthe patent's urine. These results clearly support the application of thepresent invention for the diagnosis of pathologies related to geneticmodifications.

EXAMPLE 4 DNASE ACTIVITY IN URINE

This example examines the activity of DNase present in urine byevaluating the kinetics of degradation of λ phage DNA in a urine sample.

Exogeneous λ phage DNA (200 pg) was added to 2.5 ml aliquots of a urinesample from a pregnant woman. Aliquots were incubated at 37° C. forvarious periods of time (from 0 to 2 hours), DNA was isolated andamplified by PCR (with annealing at 58° C.), as described in Example 3,to detect the presence of a λ phage DNA sequence of 200 base pairs. Thefollowing primers were used to amplify a phage lambda DNA fragment from20722 to 20921 nucleotides:

5′ CAACGAGAAAGGGGATAGTGC (SEQ ID NO: 9)

5′ AAGCGGTGTTCGCAATCTGG (SEQ ID NO: 10).

The results appear in FIG. 6. Lane 1, the positive control (200 pg of λphage DNA added to PCR tube) displays a clear band at approximately 200base pairs; Lanes 2-5, representing samples incubated for 0, 30 min., 60min. and 120 min., respectively, display sequentially decreasingsignals. The degradation activity of DNase present in the urine isapparent from this figure. In one embodiment, the present inventionencompasses the use of various methods known to one skilled in the artto prevent the degradation of DNA by DNase or other constituents ofurine.

EXAMPLE 5 Urine DNA Concentration and Purification

Various methods were tested for concentrating and purifying urinesamples to improve the sensitivity and accuracy of urine DNA detection.

Butenol Concentration. Nick-translated [³²P]labeled DNA, intact ordenatured, was added to 20-ml samples of urine and subjected to severalsteps of butanol concentration. After each concentration step, thesample volume and radioactivity of 50 μL aliquots were measured. Resultsshowed a greater than 90% reduction in sample volume over 5 extractions,with an increase of radiation of approximately 100% in the 50 μLaliquot.

Sephadex Purification. Measured amounts of dry Sephadex G-25 Coarse(Pharmacia Biotech, Inc., Piscataway N.J.), (between 2 and 4 grams,inclusive) were added to 10-ml urine samples supplemented with DextranBlue (A630 -0.1) of 200,000 Daltons. After approximately 30 minutesswelling, the void volume was removed from the mixture by filtrationunder pressure and measured. The concentration value was determined bymeasurement of Dextran Blue absorbance at 630 nm. As the amount ofSephadex increased, the void volume fell to less than 2 ml and theconcentration value increased approximately 4.5 times its originalvalue.

Isolation Of Native And Denatured DNA By Glassmilk Adsorption. Glassmilkadsorption was also tested. Native or denatured nick-translated[32P]labeled DNA was added to 2 ml urine samples and subjected toisolation by adsorption on glass powder in the presence of 6 M guanidineisothiocyanate (GITC). The adsorbent was subsequently washed with GITC,followed by a wash with isopropanol. Then, the DNA was recovered in TE.Between 80 and 90% of the DNA, both native and denatured, was recoveredby this method. It was noted that the use of ionic detergents such asEDTA, that can be used to protect DNA from DNAse activity, can also havean adverse effect on the adsorption process of some materials, includingglass beads. Thus, the samples were not treated with EDTA prior toglassmilk adsorption.

EXAMPLE 6 Detection of DNA in Urine by Hybridization

This example evaluates hybridization as a technique for DNA detectionfor use in methods of the present invention. Urine samples werecollected from pregnant women.

DNA samples isolated from 1 ml of urine were blotted onto Zeta-Probemembrane (Bio-Rad, Calif.) in 0.4 M NaOH, 10 mM EDTA using a Bio-Dot SFmicrofiltration apparatus (Bio-Rad). Pre-hybridization and hybridizationprocedures were performed by incubation in formamide based hybridizationsolution at 42° C. for 16 hours as previously described (Sambrook etal., 1989). A DNA fragment of 979 base pairs (DYZ1 -p) amplified by PCRfrom a Y Chromosome specific repeated DYZ1 sequence was used as a probefor hybridization. Novel PCR primers designed to amplify this fragmentare as follows:

L1: 5′-CCAATCCCATCCAATCCAATCTAC (SEQ ID NO: 11)

L2: 5′-GCAACGCAATAAAATGGCATGG (SEQ ID NO: 12)

DNA probes were labeled with [α-³²P] dCTP, by random priming, to aspecific radioactivity of over 5×10⁸ counts per minute (cpm)/μg.Hybridization membranes were washed with 2×SSC, 0.1% SDS at roomtemperature twice, followed by two high stringency washes with 0.1×SSC,0.1% SDS at 65° C. Kodak X-Omat AR film, with Fisher Biotech L-Plusintensifying screen, was exposed to the filters at room temperature for2-16 hours.

The results appear in FIG. 7. The negative control (lane 1, non-pregnantfemale) shows no signal; the positive control (lane 2, male total genomeDNA, 5 ng) displays hybridization; the urine samples from women carryingmale fetuses (lanes 3,4) have marked signals; and the urine samples fromwomen carrying female fetuses (lanes 5, 6) show significantly lowersignal, easily distinguished from the positive test and control samples.The faint band in lanes 6 and 7 may be a result of contaminating DNAfrom the surroundings. In the figure, clear bands appear only in thepositive control and the urine from pregnant women carrying malefetuses. Thus, hybridization is an effective technique for detection ofDNA in urine for methods of the present invention, such as the detectionof specific nucleic acid sequences that have crossed the kidney barrier,and more specifically, the determination of fetal sex. Whilehybridization technique indicates a clear distinction between sampleswhich are positive and negative for the presence of the target sequenceof DNA, the methods of the present invention also encompass theapplication of techniques to control the introduction of contaminatingDNA into the samples prior to hybridization or amplification.

EXAMPLE 7 Early Prenatal Sex Detection

This example investigates the feasibility of detecting fetal DNA inmaternal urine at early gestational ages. It is known that fetal cellsappear in maternal blood at gestational ages as early as 5-9 weeks(Eggling et al., “Determination of the origin of single nucleated cellsin maternal circulation by means of random PCR and a set of lengthpolymorphisms,” (1997) Hum. Genet. 99, 266-270; Thomas et al., “The timeof appearance, and quantitation, of fetal DNA in the maternalcirculation,” (1994) Annals NY Ac. Sci 731, 217-225). One can suggestthat apoptosis is especially active at early stages of embryonicdevelopment and, hence, an enhanced input of degraded DNA into maternalcirculation can be expected at that time, making such early detectionpossible.

To this end, pregnant women attending an antenatal clinic to havedeliberate abortion (gestation ages of 5-12 weeks) were investigatedwith informed consent. Fresh urine samples taken just prior to operationas well as samples of embryonic tissues removed during surgery werecollected.

Urine DNA was prepared for PCR amplification by two methods-simple urinedilution or adsorption onto anion exchanger DEAE-Sephadex A-25 (ParmaciaBiotech, Inc. Piscataway, N.J.). The simple urine dilution samples were10-fold diluted with distilled water, heated in a boiling bath and usedfor PCR (5-10 μl per tube, i.e., 0.5-1 μl of original urine).DEAE-Sephadex A-25 purification was performed as follows. A small volumeof urine (1-1.5 ml) was passed through a DEAE-Sephadex A-column (10-mlvolume) to remove impurities and salts. The eluate samples obtained weretaken directly to PCR. The concentration of urine DNA obtained byadsorption on anion exchanger DEAE-Sephadex A-25 appeared to besignificantly higher (approximately 500-700 ng/ml) than that estimatedpreviously (2-20 ng/ml). DNA concentration was determined byspectrofluorometry with Hoechst 33258.

PCR was performed as set forth in Example 3, above, with the followingprimers. Fetal sex was determined by PCR analysis of DNA from fetaltissue with Y1 (SEQ ID NO:1) and Y2 (SEQ ID NO:2) primers to amplify a154 base pair Y-specific DYZ1 sequence. Because the amount of fetal DNAwas sufficient, it was not necessary to perform nested PCR. Nested PCRwas carried out with maternal urine DNA samples additionally using nY1and nY2 primers to target a 77 base pair sequence found within the 154base pair sequence:

nY1: 5′-GTCCATTACACTACATTCCC-3′ (SEQ ID NO:13)

nY2: 5′-AATGCAAGCGAAAGGAAAGG-3′ (SEQ ID NO:14).

The results appear in FIG. 8. In 2 out of 5 male fetuses, Y-specificsequences were detected in maternal urine DNA at gestational ages of 7-8weeks.

EXAMPLE 8

Prenatal Testing For Congenital Diseases The principal finding ofpermeability of the kidney barrier for substantial sized DNA moleculesopens the way for the use of maternal urine to perform completelynoninvasive prenatal diagnosis of congenital diseases. One can performsuch a noninvasive screen as follows.

First, a sample of urine is gathered from a pregnant woman. Wheredesired, polymeric DNA in the urine sample can then be isolated,purified and/or treated to prevent degradation using methods known inthe art, including, but not limited to, the methods described herein.Polymeric DNA that has crossed the kidney barrier is then amplifiedusing primers specific to known disease associated genetic anomalies, oris otherwise treated to produce a detectable signal if the specificanomaly is present. Finally, the product of DNA amplification, or thesignal produced, is analyzed to determine whether or not a diseaseassociated anomaly is present in the urine DNA. Where such an anomaly isdetected and the mother does not carry the anomaly in her genome, it canbe deduced that the fetus carries the anomaly.

EXAMPLE 9 Filter Transfer of DNA

As shown in the above examples, nested PCR permits detection of smallamounts of DNA in urine. Thus, it was desired to determine whether DNAcould be analyzed directly from the urine, rather than having to performa DNA isolation step prior to amplification or other detection methods.

Female urine samples were collected (approximately 20 ml each) andtreated with several concentrations of male DNA. 3.5 cm. Hybond Nfilters (Amersham) were pretreated with 0.25 N HCl for 1.5 hours toremove any contaminating DNA, followed by rinsing with distilled water.Two filters were immersed in each urine sample and allowed to incubateovernight at room temperature with gentle shaking. The filters were thenremoved and rinsed with distilled water. DNA was desorbed by incubationof the filters with 450 μl 0.25×PCR buffer in a boiling bath for 10minutes. An aliquot (5-10 μl, i.e. 0.5-1.0 μl of original urine sample)was taken from each sample for nested PCR. The results appear in FIGS. 9and 10. In FIG. 9, lanes 1-4 represent 20 fg, 1 pg, 2 pg, and 10 pg maleDNA, respectively, per 1 μl of female urine. The Control contained 10 μlaliquots of 10-fold diluted urine, taken directly into PCR tubes. Otherurine samples were made highly concentrated in salt (10×SSC) or alkaline(adjusted to pH 12 with NaOH) and handled with the “filter transfer”method described herein. nc—negative control; m—molecular weightstandard. It is clear from the figure that the simple filter transfermethod provides a stronger signal than can be detected from a transferof an equivalent amount of urine.

Additionally, as FIG. 10 shows, adsorption of urine DNA on Hybond Nfilters appears to have protected the DNA from nuclease digestion. Thisprotection was complimented by increasing the pH of the sample. SectionA represents the controls (lanes: 1-10 μl aliquot of 10-fold dilutedurine was taken directly into PCR tubes just after male DNA addition;2-10μl aliquot of 10-fold diluted urine was taken directly into PCRtubes after incubation overnight at room temperature; 3—Hybond N filterwas incubated in urine overnight and used for DNA crossover (10 μlaliquot from eluate was used for the analysis). Section B—all theprocedures as in A, except the urine samples were made 10 mM in EDTA.Section C—all the procedures as in A, except the urine samples were made10 mM in EDTA and adjusted to pH 12.

EXAMPLE 10 Tumor Diagnostics

The ability to isolate significant quantities of DNA from urine samples,as shown in Example 3, also introduces the ability to evaluate apatient, in a non-invasive fashion, for the presence of one or more ofnumerous DNA anomalies that indicate the existence of or the propensityfor a disease of interest. Such a method has applications for the earlydiagnosis and treatment of many cancers and pathogen infections that arenot characterized by shedding of cells directly into the urinary tract,such as, but not limited to, cancers or infections that exist inisolated areas of the body and are not easily detectable by other means.One can perform such a noninvasive screen as follows.

First, a sample of urine is gathered from a patient. Where desired,polymeric DNA in the urine sample can then be isolated, purified and/ortreated to prevent degradation using methods known in the art,including, but not limited to, the methods described herein. PolymericDNA that has crossed the kidney barrier is then amplified using primersspecific to known disease associated genetic anomalies, or is otherwisetreated to produce a detectable signal if the specific anomaly ispresent.

Some methods of amplification result in improved specificity whenapplied to detect small changes in DNA, such as point mutations. Forexample, highly sensitive PCR double RFLP method (PCR-dRFLP) (Grau andGriffais, NAR, 1994, 22, 5773-5774) can be used to diagnose a mutationthat creates or destroys a natural or artificial restriction site.However, PCR-RFLP sometimes presents a technical difficulty because adefective restriction enzyme activity can be confused with the loss ofthe restriction site. Moreover, the presence of a restriction site canbe experimentally easier to ascertain than its absence. To overcome thisdifficulty, two modified nested PCR amplifications can additionally beperformed for each studied DNA, one pair of PCR primers being designedto introduce a restriction site specific for the wild-type allele whilethe second pair of primers being designed to introduce a restrictionsite specific for the mutant allele. Each PCR product is then analyzedby RFLP. These two RFLP allow a less ambiguous interpretation of theresults. Essentially, it is as though each mutation abolishes arestriction site on the wild type sequence to create a new one on themutant one.

Finally, the product of DNA amplification, or the signal produced, isanalyzed to determine whether or not a disease associated anomaly ispresent in the urine DNA, thereby permitting non-invasive diagnosis ofmany diseases characterized by modification of a patient's DNA.

EXAMPLE 11 Further Tumor Diagnostics

In this example, K-ras mutations were detected in the urine of patientswith colon adenocarcinomas and pancreatic carcinomas.

Samples of colon cancer and surrounding “normal” tissues were obtainedfrom patients undergoing surgery. Urine samples were obtained 24 hrbefore surgery.

Samples (25-50 ml) were collected fresh (i.e., accumulated duringmorning hours). The first voiding of the day was not used. The sampleswere adjusted to 10 mM EDTA and stored frozen before use. To control apotential contamination of solutions and final probes with exogeneousDNA the experimental setup also included a control (25 ml of salinesolution) that was carried through all subsequent procedures.

DNA was purified from non-fractionated urine samples (i.e., notsubjected to centrifugation) to avoid possible DNA losses due toadsorption to particulate material. Urine samples (3-5 ml) were mixed1:1.5 (v:v) with 6 M guanidine isothiocyanate and DNA was adsorbed on aWizard column (Minipreps DNA purification system, Promega), asrecommended by the manufacturer. Columns were washed with 50%isopropanol and DNA was eluted with 200 μl of distilled water.

K-ras mutations were detected by a two-stage PCR assay using selectiverestriction enzyme digestions of an artificially created site to enrichfor mutant K-ras DNA. Kopreski MS, et al., Detection of mutant K-ras DNAin plasma or serum of patients with colorectal cancer, Br. J. Cancer1997; 76:1293-99. PCR was performed with oligonucleotide primers K-ras-L(5′-ACTGAATATAAACTTGTGGTAGTTGGACCT-3′) (SEQ ID NO: 15) and K-ras-R(5′-TCAAAGAATGGTCCTGGACC-3′) (SEQ ID NO: 16). The first primer, which isimmediately upstream of codon 12, is modified at nucleotide 28 (G to C)to create an artificial restriction enzyme site (Bst NI). Theoligonucleotide K-ras-R is also modified at the base 17 (C to G) tocreate an artificial Bst NI site to serve as an internal control forcompletion of the digestion. As a result, non-restricted PCR product is157 base pairs (bp) long, while being restricted at both sites (wildtype sequence) becomes 113 bp long and restricted only at right site(the left site is modified by mutation) becomes 142 bp long. Thereaction mixture is cycled 15 times at 94° C. for 48 sec., 56° C. for 90sec., and 72° C. for 155 sec. An aliquot of 10 μl adjusted to 1×Bst NIreaction buffer was digested with 10 units of Bst NI at 60° C. for 90min. Ten μl of the digested PCR mixture was removed to a new tube and anew reaction mixture was set up for the second amplification step (35cycles—94° C. for 48 sec., 56° C. for 90 sec., and 72° C. for 48 sec.)using identical constituents. A second Bst NI restriction digestion wasperformed using 25 μl of the second-step PCR product at 60° C. for 90min. The final digestion product was separated by electrophoresisthrough a 3% Nu-Sieve agarose gel or 12% polyacrylamide gel.

Fresh urine samples were centrifuged 10 min at 800×g and DNA wasisolated from the supernatant as described above. DNA samples werestained with 0.1 μg/ml of Hoechst 33258 and DNA concentration wasdetermined by spectrofluorometry as described. Labarca C. and Paigen K.A simple, rapid, and sensitive DNA assay procedure. Analyt Biochem 1980;102: 344-52.

Two groups of patients were analyzed for K-ras mutations in their urineDNA. It is known that 80-90% of pancreatic carcinomas show K-rasmutations. The first group consisted of 8 patients with pancreaticcancer (stage IV). K-ras mutations were detected in 5 of the 8 urinesamples from these patients.

The second group consisted of 7 colorectal cancer patients with advanceddisease (stages III-IV). Urine samples were taken 24 hr before surgeryand tissue samples, tumor and surrounding normal tissue, were obtainedduring surgery. Thus, three samples obtained from each patient, tumor,normal tissue and urine DNA, were analyzed. K-ras mutations weredetected in 5 out of 7 tumors. In 4 of 5 patients with tumor K-rasmutations, the same mutations were also detected in the urine samples.Two patients that had no mutation in the tumor and 9 healthy volunteersdid not show K-ras mutations in their urine DNA.

In conclusion, K-ras mutations were found in 5/8 urine samples obtainedfrom patients with pancreatic cancer as well as in urine of 4/5 patientswith colorectal adenocarcinomas that have corresponding mutation intheir tumors. The results of this experiment support that the in oneembodiment of the present invention, methods are useful for thedetection and monitoring of tumor growth, including the evaluation ofthe effectiveness of tumor chemo- or radio-therapy.

EXAMPLE 12 ANALYSIS OF DNA METHYLATION

For the analysis of DNA methylation we will use a methylation specificPCR strategy (Herman JG, et al., Methylation-specific PCR: A novel PCRassay for methylation status of CpG islands, Proc Natl Acad Sci US 1996;93: 9821-26) and its recent modification for Real Time PCR (Eads CA, etal., MethyLight: a high-throughput assay to measure DNA methylation,Nucl Acids Res 2000 28: 32e-40e). The method is based on registration ofthe sequence differences between methylated and unmethylated allelesresulting from the bisulfite treatment of DNA. The bisulfitemodification includes several steps of chemical treatments resulting inthe conversion of unmethylated cytosine residues into uracil while themethylated derivatives remain unchanged. Wang RY, et al., Comparison ofbisulfite modification of 5-methyldeoxycytidine and deoxycytidineresidues, Nucl Acids Res 1980; 8: 4777-90.

For the high resolution CpG methylation mapping we will use+ddC-sequencing reaction.

Three pairs of primers are needed for the amplification and resolutionof a methylated and unmethylated CpG island. First, primers that arespecific to the “wild” type (unconverted); second, methylated/convertedprimers, and third, unmethylated/converted primers. To increase thesensitivity of the reaction, a second set of nested PCR primers may beneeded. For the real time PCR analysis an additional DNA probe, bearingreporter dye, will be used that is specific to an amplified fragment.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications can be practiced. Therefore, thedescriptions and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

16 1 23 DNA Artificial Sequence Description of Artificial SequenceY1primer for amplification of human Y-chromosome specific DYZ1 repeatfragment 1 tccactttat tccaggcctg tcc 23 2 23 DNA Artificial SequenceDescription of Artificial SequenceY2 primer for amplification of humanY-chromosome specific DYZ1 repeat fragment 2 ttgaatggaa tgggaacgaa tgg23 3 23 DNA Artificial Sequence Description of Artificial SequenceYZ1primer for amplification of human Y-chromosome specific DYZ1 repeatfragment 3 ccattccttt gcattccgtt tcc 23 4 23 DNA Artificial SequenceDescription of Artificial SequenceYZ2 primer for amplification of humanY-chromosome specific DYZ1 repeat fragment 4 atcgactggc agggaaccaa aag23 5 20 DNA Artificial Sequence Description of Artificial SequenceY1.5primer for nested PCR of human Y-chromosome specific DYS14 single-copygene marker 5 ctagaccgca gaggcgccat 20 6 21 DNA Artificial SequenceDescription of Artificial SequenceY1.6 primer for nested PCR of humanY-chromosome specific DYS14 single-copy gene marker 6 tagtacccacgcctgctccg g 21 7 21 DNA Artificial Sequence Description of ArtificialSequenceY1.7 primer for nested PCR of human Y-chromosome specific DYS14single-copy gene marker 7 catccagagc gtccctggct t 21 8 21 DNA ArtificialSequence Description of Artificial SequenceY1.8 primer for nested PCR ofhuman Y-chromosome specific DYS14 single-copy gene marker 8 ctttccacagccacatttgt c 21 9 21 DNA Artificial Sequence Description of ArtificialSequence amplification primer for lambda phage DNA fragment 9 caacgagaaaggggatagtg c 21 10 20 DNA Artificial Sequence Description of ArtificialSequence amplification primer for lambda phage DNA fragment 10aagcggtgtt cgcaatctgg 20 11 24 DNA Artificial Sequence Description ofArtificial SequencePCR amplification primer L1 for human Y-chromosomespecific DYZ1-p repeat fragment 11 ccaatcccat ccaatccaat ctac 24 12 22DNA Artificial Sequence Description of Artificial SequencePCRamplification primer L2 for human Y-chromosome specific DYZ1-p repeatfragment 12 gcaacgcaat aaaatggcat gg 22 13 20 DNA Artificial SequenceDescription of Artificial Sequencenested PCR primer nY1 for humanY-chromosome specific DYZ1 repeat fragment 13 gtccattaca ctacattccc 2014 20 DNA Artificial Sequence Description of Artificial SequencenestedPCR primer nY2 for human Y-chromosome specific DYZ1 repeat fragment 14aatgcaagcg aaaggaaagg 20 15 30 DNA Artificial Sequence Description ofArtificial Sequencemutant K-ras PCR oligonucleotide primer K-ras-L 15actgaatata aacttgtggt agttggacct 30 16 20 DNA Artificial SequenceDescription of Artificial Sequencemutant K-ras PCR oligonucleotideprimer K-ras-R 16 tcaaagaatg gtcctggacc 20

We claim:
 1. A method of monitoring transplanted material in a patient,comprising: a) providing a urine sample suspected of containing nucleicacid of the transplanted material, which transplanted material islocated outside of the urinary tract; and b) analyzing said urine samplefor nucleic acids from the cell genome of the transplanted material thatare different from nucleic acids of the recipient and that have crossedthe kidney barrier.
 2. The method of claim 1, wherein said nucleic acidsequence is not present in cells of the urinary tract of said patient.3. The method of claim 1, wherein said analyzing comprises amplifyingsaid nucleic acid sequence with a primer substantially complementary toa part of said nucleic acid sequence that does not occur in cells of theurinary tract of the patient, to make amplified target DNA, anddetecting the presence of said amplified target DNA.
 4. The method ofclaim 3, wherein amplifying comprises performing a polymerase chainreaction.
 5. The method of claim 1, further comprising step (a)(i)reducing DNA degradation in said urine sample.
 6. The method of claim 5,wherein reducing DNA degradation is by treatment with a compoundselected from the group consisting of: ethylenediaminetetraacetic acid,guanidine-HCl, Guanidine isothiocyanate, N-lauroylsarcosine, andNa-dodecylsulphate.
 7. The method of claim 1, wherein said urine samplehas been held in the bladder less than 12 hours.
 8. The method of claim1, further comprising step (a)(i) substantially isolating said nucleicacid sequence.
 9. The method of claim 8, wherein said nucleic acidsequence is substantially isolated by precipitation.
 10. The method ofclaim 8, wherein said nucleic acid sequence is substantially isolated byadsorption on a resin.
 11. The method of claim 1, further comprisingstep (a)(l) filtering said urine sample to remove contaminants.
 12. Themethod of claim 11, wherein said filtering removes DNA comprising morethan about 1000 nucleotides.